US8050871B2 - Method of predicting influenza outbreaks by correlating an increase in replikin count in shrimp white spot syndrome virus and/or taura syndrome virus - Google Patents

Method of predicting influenza outbreaks by correlating an increase in replikin count in shrimp white spot syndrome virus and/or taura syndrome virus Download PDF

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US8050871B2
US8050871B2 US11/923,559 US92355907A US8050871B2 US 8050871 B2 US8050871 B2 US 8050871B2 US 92355907 A US92355907 A US 92355907A US 8050871 B2 US8050871 B2 US 8050871B2
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replikin
virus
isolates
concentration
isolate
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US20080176217A1 (en
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Samuel Bogoch
Elenore S. Bogoch
Samuel Winston BOGOCH
Anne Elenore BORSANYI
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Priority to AU2008253721A priority patent/AU2008253721A1/en
Priority to JP2009546425A priority patent/JP2011516027A/en
Priority to CA002676028A priority patent/CA2676028A1/en
Priority to CN200880002494A priority patent/CN101688236A/en
Priority to KR1020097014345A priority patent/KR20090094838A/en
Priority to EP08794275A priority patent/EP2126140A4/en
Priority to PCT/US2008/061336 priority patent/WO2008156914A2/en
Priority to AU2008266702A priority patent/AU2008266702A1/en
Priority to MX2009013091A priority patent/MX2009013091A/en
Priority to US12/108,458 priority patent/US9408902B2/en
Priority to CN2008800182411A priority patent/CN101969993B/en
Priority to CA002689181A priority patent/CA2689181A1/en
Priority to EP13000747.9A priority patent/EP2594578A1/en
Priority to EP08825968A priority patent/EP2167122A2/en
Priority to KR1020097024437A priority patent/KR20100006574A/en
Priority to NZ581332A priority patent/NZ581332A/en
Priority to JP2010510390A priority patent/JP5675348B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • This invention relates generally to predicting influenza outbreaks through monitoring quantitative changes in the concentration of a class of peptide sequences known as Replikins in virus proteins, protein fragments, or genomes.
  • Rapid replication is characteristic of virulence in, among other things, certain bacteria, viruses and malignancies.
  • the inventors have described a quantitative chemistry common to rapid replication in different viruses and organisms.
  • the chemistry of rapid replication described by the inventors is present in a family of conserved small protein sequences related to rapid replication, Replikins.
  • An increase in the concentration of these Replikin sequences encoded in the genome of different strains of influenza virus has been correlated with an increase in the virulence of influenza.
  • a correlation between increased concentrations of Replikin sequences and increased replication and virulence has likewise been observed in a range of viruses and organisms.
  • Replikin sequences offer new targets for developing effective methods of predicting and treating influenza outbreaks. There continues to exist a particular need in the art for methods of predicting viral outbreaks.
  • Influenza is an acute respiratory illness of global importance. Despite international attempts to control influenza virus outbreaks through vaccination, influenza infections remain an important cause of morbidity and mortality. Worldwide influenza pandemics have occurred at irregular and previously unpredictable intervals throughout history and it is expected that influenza pandemics will continue to occur in the future. The impact of pandemic influenza is substantial in terms of morbidity, mortality and economic cost.
  • Influenza vaccines remain the most effective defense against influenza virus, but because of the ability of the virus to mutate, and the availability of non-human host reservoirs, it is expected that influenza will remain an emergent or re-emergent infection.
  • Global influenza surveillance indicates that influenza viruses may vary within a country and between countries and continents during an influenza season. Virologic surveillance is of importance in monitoring antigenic shift and drift. Disease surveillance is also important in assessing the impact of epidemics. Both types of information have provided the basis of vaccine composition and use of antivirals.
  • the only basis for annual classification of influenza virus as active, inactive or prevalent in a given year was the activities of the virus hemagglutinin and neuraminidase proteins.
  • a Replikin sequence is an amino acid sequence of 7 to about 50 amino acids comprising a Replikin motif.
  • a Replikin motif comprises (1) at least one lysine residue located at a first terminus of the motif and at least one lysine residue or at least one histidine residue located at a second terminus of the motif, (2) a first lysine residue located six to ten residues from a second lysine residue; (3) at least one histidine residue; and (4) at least 6% lysine residues.
  • a Replikin sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine.
  • a Replikin peptide or Replikin protein is a peptide or protein consisting of a Replikin sequence.
  • Replikin sequences in the genomic code are associated with a variety of infectious agents including HIV, plant viruses, and a range of pathogenic animal and human viruses including flu viruses. Further, the correlation between the concentration of Replikin sequences in viral or organismal proteins and major outbreaks of disease is significant. Replikin sequences generally have been found to be conserved in both intrastrain and interstrain influenza viruses for as long as 89 years based on data going back to the 1917-18 flu pandemic. Concentration of Replikin sequences in viral genomes has been shown to increase prior to strain-specific flu outbreaks.
  • Replikin sequences The highest concentration of Replikin sequences in an organism or virus that has to date been analyzed and reported is 111 Replikin sequences per 100 amino acids in the extraordinarily-rapidly-replicating parasitic protozoa Plasmodium falciparum (reportedly responsible for 90% of malarial deaths in humans) (herein sometimes referred to as malaria). P. falciparum has been observed to replicate 11,000 times in 48 hours during passage of the parasite from liver to blood in the host.
  • Replikin Scaffolds In monitoring Replikin sequences in influenza virus, the inventors have additionally identified a sub-family of conserved Replikin sequences known as Replikin Scaffolds or Replikin Scaffold sequences.
  • Replikin Scaffolds were initially identified in conserved structures in particularly virulent influenza viruses. Included among these strains were the viruses causing the pandemics of 1918, 1957, 1968 and virulent strains of the H5N1 “bird flu” strain of influenza virus.
  • Analogues of Replikin Scaffold sequences have since been identified in the virulent and rapidly replicating SARS coronavirus. See U.S. Published Application No. 2007/0026009.
  • Replikin scaffolding in general has been related to an increase in Replikin concentrations in pathogenic genomes where it has been identified. In P. falciparum , scaffolding contributes significantly to the very high Replikin concentration noted in the proteins of the protozoa.
  • the present invention provides a method of determining an increased probability of an outbreak of influenza virus within about one to about three years following an increase in Replikin concentration in an isolate of White Spot Syndrome Virus or an isolate of Taura Syndrome Virus comprising identifying said increase in the concentration of Replikin sequences in at least one first isolate of White Spot Syndrome Virus or Taura Syndrome Virus as compared to at least one other isolate of White Spot Syndrome Virus or Taura Syndrome Virus wherein said at least one first isolate is isolated at least six months later than said at least one other isolate is isolated and wherein when said at least one first isolate is a White Spot Syndrome Virus isolate said at least one other isolate is a White Spot Syndrome Virus isolate and when said at least one first isolate is a Taura Syndrome Virus isolate said at least one other isolate is a Taura Syndrome Virus isolate, and wherein said increase in the concentration of Replikin sequences signifies the increased probability of the outbreak of influenza virus within about one to about three years following said increase in the concentration of Replikin sequences.
  • said identification of an increase in Replikin concentration in said at least one first isolate comprises (1) determining the concentration of Replikin sequences in (i) a plurality of isolates of White Spot Syndrome Virus wherein said at least one first isolate has been isolated about six months to about three years later than said at least one other of said isolates of White Spot Syndrome Virus, or (ii) a plurality of isolates of Taura Syndrome Virus wherein said at least one first isolate of Taura Syndrome Virus is isolated about six months to about three years later than at least one other isolate of Taura Syndrome Virus.
  • the increase in concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus or in said plurality of isolates of Taura Syndrome Virus over at least one time period of about six months or greater is an increase in the mean concentration of at least two isolates as compared to at least two other isolates isolated at a later time point of about six months or greater.
  • the increase in concentration is an increase in the mean concentration of at least 10 isolates as compared to at least 10 other isolates.
  • the increase in concentration is an increase in the mean concentration of all isolates available at a given time point as compared to all isolates available at another given time point, wherein said time points are separated by about six months or greater.
  • the mean concentration of said plurality of isolates is increased over the at least one time period when the mean concentration of the isolates at a later time point is greater than the mean concentration plus one standard deviation of the earlier isolates. In a further embodiment, the mean concentration of said plurality of isolates is increased over the at least one time period when the mean concentration of the isolates at a later time point is greater than the mean concentration plus two standard deviations of the earlier isolates.
  • analyzing the combination of encoded proteins and/or protein fragments preferably comprises all amino acid sequences available for White Spot Syndrome Virus or all amino acid sequences available for Taura Syndrome Virus at a given time point, such as, for example, all amino acid sequences available in a first year and all amino acid sequences available in some other year.
  • the combination comprises at least 100 amino acid sequences.
  • the combination comprsises at least 50 amino acid sequences.
  • the combination comprises at least 10 amino acid sequences.
  • the combination comprises at least 2 amino acid sequences.
  • the method of predicting the outbreak of influenza virus may further comprise:
  • the Replikin Scaffold is a peptide having about 27 to about 33 amino acids.
  • a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a preferred embodiment about 28 to about 30 amino acid residues.
  • a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a preferred embodiment about 29 to about 31 amino acid residues.
  • a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a preferred embodiment about 29 to about 33 amino acid residues.
  • the method of predicting an outbreak of influenza virus may comprise the prediction of an outbreak of any strain of influenza virus.
  • the method of predicting an outbreak of influenza virus may comprise the prediction of an outbreak of influenza A.
  • the outbreak of influenza A may be an outbreak of H5N1 (“bird flu”) virus.
  • a plurality of isolates of White Spot Syndrome Virus may be obtained and analyzed for Replikin concentration or a plurality of isolates of Taura Syndrome Virus may be obtained and analyzed for Replikin concentration, or both in order to predict an outbreak of influenza virus.
  • the concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus or said plurality of isolates of Taura Syndrome Virus over at least one time period of about six months or greater may increase by one or more Replikin sequences per 100 amino acids, may increase by five or more Replikin sequences per 100 amino acids, may increase by ten or more Replikin sequences per 100 amino acids, may increase by fifty or more Replikin sequences per 100 amino acids, may increase by 90 or more Replikin sequences per 100 amino acids, or may increase by 100 or more Replikin sequences per 100 amino acids.
  • the identified increase in concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus or said plurality of isolates of Taura Syndrome Virus may occur over any time period including over one month or more, over six months or more, over one year or more, or over three years or more.
  • the identified increase in concentration of encoded Replikin sequences occurs over at least six months to about five years.
  • the identified increase in concentration of encoded Replikin sequences preferably occurs over at least six months to about three years.
  • the method of predicting an outbreak of influenza may predict an outbreak within about one month to about five years or more following the identification of an increase in Replikin concentration in a plurality of isolates of White Spot Syndrome Virus or in a plurality of isolates of Taura Syndrome Virus. In a further embodiment of the invention, the method may predict an outbreak within several months to about three years.
  • the method may predict an outbreak within about one year to about five years. In a further embodiment of the invention, the method may predict an outbreak within several months to about one year. In another embodiment, the method may predict an outbreak within about one year to about three years.
  • Another aspect of the invention provides a method of predicting an outbreak of influenza virus comprising analyzing Replikin concentration in reservoirs for influenza virus.
  • a non-limiting embodiment of the invention provides a method of predicting an outbreak of influenza virus comprising:
  • the reservoir is shrimp.
  • the reservoir is a bird.
  • the reservoir is a migratory bird.
  • the reservoir is a chicken, duck, goose, or other domestic bird.
  • the method of predicting an outbreak of influenza virus further comprises:
  • FIG. 1 illustrates a correlation between (1) the mean concentration and standard deviation of Replikin sequences observed in amino acid sequences of White Spot Syndrome Virus (WSSV) isolated between 1995 and 2007 that have publicly available accession numbers at www.pubmed.com, (2) the mean concentration of Replikin sequences in the pB1 gene area of H5N1 strains of influenza virus isolated in 2003, 2004, 2005, 2006, and 2007 that have publicly available accession numbers at www.pubmed.com, and (3) the mean mortality rate for humans infected with H5N1 influenza virus isolated in 2005, 2006 and 2007.
  • the data demonstrate an exceptionally high Replikin sequence concentration in WSSV in 2000 followed within three years by an increase in Replikin sequence concentration in H5N1 isolates (from 2003 through 2007) and a concomitant outbreak of H5N1 influenza in humans with rising mortality.
  • FIG. 2 illustrates a correlation between (1) the mean concentration and standard deviation of Replikin sequences observed in Taura Syndrome Virus (TSV) isolated between 2000 and 2005 that have publicly available accession numbers at www.pubmed.com, (2) the mean concentration of Replikin sequences in the pB1 gene area of H5N1 strains of influenza virus isolated in 2003, 2004, 2005, 2006, and 2007 that have publicly available accession numbers at www.pubmed.com, and (3) the mean mortality rate for humans infected with H5N1 influenza virus isolated in 2005, 2006, and 2007.
  • TSV Taura Syndrome Virus
  • FIG. 3 illustrates a correlation between Replikin concentration in the H5N1 strain of influenza virus and human mortality from 2005 through the first quarter of 2007.
  • FIG. 3 contains a graph comparing percent human mortality from H5N1 infections in years 2005 through the first quarter of 2007, mean concentration of Replikin sequences in the genome of H5N1 influenza strains isolated in 2003 through the first quarter of 2007 and mean concentration of Replikin sequences in the pB1 gene area of H5N1 influenza strains isolated in 2003 through the first quarter of 2007.
  • Mean human mortality in deaths per 10 infections in a given year is represented by the black columns
  • mean Replikin concentration in the whole virus genome in a given year is represented by the light gray columns
  • mean Replikin concentration in the pB1 gene area in a given year is represented by the dark gray columns
  • standard deviations are represented by the white columns.
  • FIG. 4 illustrates that an increasing Replikin concentration of hemagglutinin protein in the H5N1 strain of influenza virus preceded three “Bird Flu” Epidemics.
  • the decline in 1999 occurred with the massive culling of poultry in response to the E 1 epidemic in Hong Kong.
  • FIG. 5 illustrates a direct sequential correlation between Replikin concentration in isolates of Taura Syndrome Virus (TSV) collected from Caribbean, Thailand, Hawaii and Venezuela, respectively, and mean number of days until 50% mortality in Litopenaeus vannamei shrimp challenged with the respective TSV isolates on day zero. Statistical differences between the Replikin concentration for each isolate are significant at a level of p ⁇ 0.001.
  • TSV Taura Syndrome Virus
  • FIG. 6 illustrates a direct sequential correlation between Replikin concentration in isolates of Taura Syndrome Virus (TSV) collected from Caribbean, Thailand, Hawaii and Venezuela, respectively, and mean cumulative survival of Litopenaeus vannamei shrimp at 15 days after challenge with the respective TSV isolate. Statistical differences between the Replikin concentration for each isolate are significant at a level of p ⁇ 0.001.
  • TSV Taura Syndrome Virus
  • FIG. 7 illustrates that cumulative survival of Litopenaeus vannamei shrimp challenged with four different Taura Syndrome Virus isolates over 15 days (unless 100% mortality occurred prior to 15 days) correlates sequentially with the Replikin concentration of Open Reading Frame 1 (ORF1) of each isolate.
  • ORF1 Open Reading Frame 1
  • Replikin concentration was determined to be 3.5 for the Caribbean isolate, 3.4 for the Thailand isolate, 3.3 for the Hawaii isolate and 3.0 for the Venezuela isolate.
  • Graph A illustrates observed percent survival in three trials of shrimp challenged with the Caribbean isolate of TSV. In one trial, total mortality was observed on day 6. In the other two trials, total mortality was observed on day 11.
  • Graphs B, C and D illustrate observed percent survival of shrimp challenged with the Thailand isolate, the Hawaii isolate and the Venezuela isolate, respectively, each in three trials over 15 days.
  • Thailand isolate a mean of 80% percent mortality was observed on day 15.
  • Hawaii isolate a mean of 78.3% mortality was observed on day 15.
  • Venezuela isolate a mean of 58.3% mortality was observed on day 15.
  • FIG. 8 illustrates the localization of the pB1 gene area as the Replikin Peak Gene in the genome of the H5N1 strain of influenza virus.
  • FIG. 8 discloses a bar graph depicting the number (with standard deviation) of Replikins per 100 amino acids in the eight genes of H5N1 influenza virus identified annually in humans between 2003 and 2006. A significant increase is observed in the pB1 gene area as compared to the other seven genes of the H5N1 influenza virus strain.
  • FIG. 9 illustrates a correlation between an increase in Replikin concentration in the pB1 gene area (Replikin Peak Gene) of the H5N1 strain of influenza virus in various bird species and humans over time with an increase in virulence of the H5N1 strain of influenza.
  • FIG. 9 discloses a graph depicting the number (with standard deviation) of Replikins per 100 amino acids in the pB1 gene area (Replikin Peak Gene) of H5N1 influenza virus strains identified annually in duck and chicken in years 2001 through 2006, in goose in years 2001 and 2003 through 2006, and in human in years 2003 through 2006.
  • FIG. 10 is a bar graph depicting the number (with standard deviation) of Replikins per 100 amino acids in the pB1 gene area (Replikin Peak Gene) of H5N1 influenza virus strains identified annually in humans in Japan, Russia, Egypt, China, Vietnam, Thailand and Indonesia between 2003 and 2006. Incidence of human morbidity and mortality in the Indonesian outbreak were exceptionally high and evidence of possible human to human transmission was observed. Changes in Replikin concentration in the Replikin Peak Gene of the H5N1 isolates in FIG. 10 allows for identification of those geographic areas in which the influenza virus strain is more virulent than other geographic areas.
  • FIG. 11 is a graph correlating Replikin Count in each of three strains of influenza virus responsible for one of three pandemics during the 20 th century with virulence.
  • each pandemic being strain specific: H1N1 in 1918; H2N2 in 1957; and H3N2 in 1968.
  • the x-axis of FIG. 11 shows the year and the y-axis shows the Replikin concentration in Replikin sequences per 100 amino acids. Note that within FIG. 11 there is a separate graph for each strain responsible for each pandemic.
  • FIG. 12 is a graph illustrating an increase in Replikin concentration in spike and nucleocapsid coronavirus proteins preceding the SARS coronavirus epidemic of 2003.
  • the x-axis indicates the year and the y-axis indicates the Replikin concentration.
  • the appearance of the SARS outbreak and the eight countries involved in the outbreak is shown by the conical shaded area.
  • the solid black symbols represent the mean Replikin concentration for spike coronavirus proteins and the vertical black bars represent the standard deviation of the mean.
  • FIG. 12 illustrates a remarkable constancy of low coronavirus Replikin concentration between 1995 and 2001 in the spike proteins, followed by a dramatic increase in 2002, one year before the SARS epidemic appeared in 2003.
  • FIG. 13 is a graph comparing (1) the concentration of Replikin sequences observed in isolates of West Nile Virus having publicly available accession numbers on a year by year basis between 1999 and 2006 (with standard deviation bars for each Replikin concentration data point except 2000), (2) total morbidity reported in the United States on a year by year basis by the Center for Disease Control (total U.S. morbidity is the value denoted on the y-axis times 1000), and (3) total mortality resulting from WNV infection reported in the United States on a year by year basis by the Center for Disease Control (total U.S. mortality is the value denoted on the y-axis times 100).
  • a correlation between Replikin concentration, morbidity and mortality is demonstrated.
  • FIG. 14 is a graph illustrating the mean concentration of Replikins observed in the VP1 protein of isolates of Foot and Mouth Disease Virus having publicly available accession numbers on a year by year basis between 1998 and 2006. Observed outbreaks of Foot and Mouth Disease in the United Kingdom (UK) are noted in 2001 and 2007.
  • FIG. 15 illustrates an increase in Replikin concentration in a Replikin Peak Gene in the pB1 genomic area of Equine Influenza one to two years in advance of epidemics of Equine Influenza.
  • the graph illustrates that no increase in Replikin concentration in pA or pB2 genes of the equine influenza virus correlates with the increase in the Replikin concentration of the Replikin Peak Gene or with outbreaks of the virus.
  • FIG. 16 illustrates a magnification of the effect of increases in Replikin concentration on human mortality from H5N1 infections when Replikin concentration is observed in the Replikin Peak Gene (pB1 gene area) as compared to the polymerase gene or compared to the entire genome of the H5N1 virus.
  • FIG. 16 is a graph of the data disclosed in Table 10. The graph demonstrates that an increase in Replikin concentration is magnified in its correlation with human mortality when restricted from changes in Replikin concentration in the entire genome to changes in Replikin concentration in the polymerase gene and magnified even more when restricted solely to the Replikin Peak Gene identified using the methods described herein.
  • FIG. 10 illustrates a magnification of the effect of increases in Replikin concentration on human mortality from H5N1 infections when Replikin concentration is observed in the Replikin Peak Gene (pB1 gene area) as compared to the polymerase gene or compared to the entire genome of the H5N1 virus.
  • FIG. 16 is a graph of the data disclosed in Table 10. The
  • FIG. 17 illustrates a significant eight-fold increase in Replikin concentration in the pB1 gene area (Replikin Peak Gene) of isolates of H5N1 from 2003 through the first quarter of 2007 (that correlates with an increase in host mortality in humans), while no significant increase is observed in neighboring gene areas of the pB1 gene area, namely, the pA gene area and the pB2 gene area.
  • FIG. 17 graphically compares percent human mortality from H5N1 infections in years 2005 through the first quarter of 2007 to mean concentration of Replikin sequences in (1) the pB1 gene area, (2) the pB2 gene area, and (3) the pA gene area, respectively, of H5N1 influenza strains isolated in 2003 through the first quarter of 2007.
  • FIG. 17 graphically compares percent human mortality from H5N1 infections in years 2005 through the first quarter of 2007 to mean concentration of Replikin sequences in (1) the pB1 gene area, (2) the pB2 gene area, and (3) the pA gene area, respectively, of H5N1 influenza
  • FIG. 17 illustrates a significant correlation between human mortality and the Replikin Peak Gene (pB1 gene area) of isolates of H5N1 influenza virus. No correlation is observed in neighboring gene areas of the pB1 gene area.
  • FIG. 17 (graphically representing part of the data in Table 10) provides strong confirmation of the power and validity of the methodology of predicting changes in virulence and outbreaks of virus by monitoring changes in Replikin concentration.
  • animal includes mammals, such as humans.
  • peptide or “protein” refers to a compound of two or more amino acids in which the carboxyl group of one amino acid is attached to an amino group of another amino acid via a peptide bond.
  • isolated or “synthesized” peptide or biologically active portion thereof refers to a peptide that is, after purification, substantially free of cellular material or other contaminating proteins or peptides from the cell or tissue source from which the peptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized by any method, or substantially free from contaminating peptides when synthesized by recombinant gene techniques.
  • an “encoded” protein, protein sequence, protein fragment sequence, or peptide sequence is a sequence encoded by a nucleic acid sequence that encodes the amino acids of the protein or peptide sequence with any codon known to one of ordinary skill in the art now or hereafter. It should be noted that it is well-known in the art that, due to redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon and still result in an identical amino acid sequence. As will be understood by one of skill in the art, a method of identifying a Replikin amino acid sequence also encompasses a method of identifying a nucleic acid sequence that encodes a Replikin amino acid sequence wherein the Replikin amino acid sequence is encoded by the identified nucleic acid sequence.
  • a Replikin sequence is an amino acid sequence having about 7 to about 50 amino acids comprising:
  • a Replikin sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine.
  • a Replikin peptide or Replikin protein is a peptide or protein consisting of a Replikin sequence.
  • a Replikin sequence may also be described as a Replikin sequence of about 7 to about 50 amino acids comprising or consisting of a Replikin motif wherein the Replikin motif comprises:
  • Replikin sequence can also refer to a nucleic acid sequence encoding an amino acid sequence having about 7 to about 50 amino acids comprising:
  • a Replikin Peak Gene or a Replikin Peak Gene Area (RPGA) are used interchangeably to mean a segment of a genome, protein, segment of protein, or protein fragment in which an expressed gene or gene segment has a highest concentration of continuous, non-interrupted and overlapping Replikin sequences (number of Replikin sequences per 100 amino acids) when compared to other segments or named genes of the genome.
  • the gene or gene segment associated with a whole protein or protein-expressing gene is known as the Replikin Peak Gene and the gene or gene segment associated with a protein fragment is known as a Replikin Peak Gene Area.
  • More than one RPG or RPGA may be identified within a gene, gene segment, protein, or protein fragment.
  • An RPG or RPGA may have a terminal lysine or a terminal histidine, two terminal lysines, or a terminal lysine and a terminal histidine.
  • An RPG or RPGA may likewise have neither a terminal lysine or a terminal histidine as long as it contains a Replikin sequence or Replikin sequences defined by the definition of a Replikin sequence, namely, an amino acid sequence having about 7 to about 50 amino acids comprising:
  • “reservoir” is any source of Replikin sequences that may be shared with an influenza virus including any host of influenza virus, any food source of a host of the influenza virus, any vector of influenza virus, or any substance wherein the genetic information of the influenza virus may be shared, mingled, mixed, exchanged, or come into the proximity of the Replikin sequences of the reservoir.
  • “different time periods” or “different time points” is any two time periods or points that may be differentiated one from another.
  • an isolate of virus isolated during the year 2004 is isolated in a different time period than an isolate of the same virus isolated during the year 2005.
  • an isolate of virus isolated in May 2004 is isolated in a different time period than an isolate of the same virus isolated in June 2004.
  • Replikin concentrations of different isolates it is preferred to use comparable time periods for comparison.
  • an isolate from 2004 is preferably compared to at least one other isolate from some other year such as 2002 or 2005.
  • an isolate from May 2004 is preferably compared to at least one isolate from some other month of some year, for example, an isolate from December 2003 or from June 2004.
  • an “earlier-arising” virus or organism is a specimen of a virus or organism collected from a natural source of the virus or organism on a date prior to the date on which another specimen of the virus or organism was collected from a natural source.
  • a natural source includes, but is not limited to, a reservoir of a virus, a vector of a virus, or a host of the virus.
  • a “later-arising” virus or organism is a specimen of a virus or organism collected from a natural source of the virus (including, but not limited to, a reservoir, a vector, or a host) or a natural source of the organism on a date subsequent to the date on which another specimen of the virus or organism was collected from a natural source.
  • “emerging strain” refers to a strain of a virus identified as having an increased or increasing concentration of Replikin sequences in one or more of its protein sequences relative to the concentration of Replikins in other strains of such organism.
  • the increased or increasing concentration of Replikins occurs over a period of preferably at least about six months, at least about one year or at least about three years, but may be a much shorter period of time for highly mutable viruses.
  • An emerging strain of virus indicates an increase in virulence or replication.
  • bird is any avian species including migratory and domestic birds, wherein said migratory and domestic birds includes, for example, chickens, ducks of all kinds, geese, pigeons, gulls, seabirds etc.
  • outbreak is an increase in virulence, morbidity or mortality in a viral disease as compared to a baseline of an earlier occurring epidemiological pattern of infection in the same viral disease.
  • morbidity is the number of cases of a disease caused by the virus, either in excess of zero cases in the past or in excess of a baseline of endemic cases in the past. Therefore the baseline of endemic cases, in epidemiological terms, may, for example, relate to whether no or some cases were present in a geographic region in the immediate past.
  • the past, in epidemiological terms may mean more than one year and can mean several years or more as understood by one of ordinary skill in the art. The past may also mean less than one year as determined by one of ordinary skill in the art.
  • the baseline reflects an annual recurrence of common influenza.
  • mutation refers to a change in the structure and properties of a virus or organism caused by substitution of amino acids.
  • conserved refers to conservation of particular amino acids due to lack of substitution.
  • a “point mutation” may refer to a change in a single amino acid residue or may refer to a change in a small number of amino acid residues.
  • replikin count or “replikin concentration” refers to the number of Replikins per 100 amino acids in a protein, protein fragment, virus, or organism.
  • a higher Replikin concentration in a first strain of a virus or organism has been found to correlate with more rapid replication of the first virus or organism as compared to a second, earlier-arising or later-arising strain of the virus or organism having a lower Replikin concentration.
  • a “Replikin Scaffold” refers to a series of conserved Replikin peptides wherein each of said Replikin peptide sequences comprises about 16 to about 34 amino acids, and preferably about 27 to about 33 amino acids and further comprises: (1) a terminal lysine and optionally a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and optionally a histidine immediately adjacent to the terminal histidine; (3) a lysine within 6 to 10 amino acid residues from another lysine; and (4) about 6% lysine.
  • “Replikin Scaffold” also refers to an individual member or a plurality of members of a series of Replikin Scaffolds.
  • a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a preferred embodiment about 28 to about 30 amino acid residues.
  • a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a more preferred embodiment about 29 to about 31 amino acid residues.
  • a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a more preferred embodiment about 29 to about 33 amino acid residues.
  • the inventors provide herein methods of predicting outbreaks of influenza by monitoring the presence and/or concentration of Replikin sequences both throughout the virus and in specific highest concentrations of Replikins in areas designated as Replikin Peak Genes, Replikin Peak Gene Areas and/or Replikin Scaffold sequences in WSSV and TSV in shrimp and influenza viruses.
  • a Replikin Peak Gene includes the area in a genome, protein, or protein fragment that has the highest concentration of Replikin sequences. While increased concentration of Replikin sequences in the genome of a virus offers both advance warning and new targets for developing effective methods of predicting and treating viral outbreaks, identification of an increase in concentration of Replikin sequences in a Replikin Peak Gene of a genome or protein heightens the predictive capacity of the change in Replikin concentration and the efficacy of new targets.
  • emerging viral diseases can be identified in virus reservoirs and vectors in advance of their appearance in animal or human hosts. Identification of the emerging viruses and the Replikin sequences within the virus genome allows for appropriate, advance control efforts, including isolation and quarantine, and provides sufficient time for the synthesis and testing of vaccines specific to the sequences of the emerging virus.
  • Replikin sequences as infectious units (and providing particular areas in the genome, i.e. Replikin Peak Genes, where those infectious units are correlated most significantly with virulence) has allowed the inventors to focus attention on the geographic area (see FIG. 10 ), the host (see FIG. 9 ), and the particular area of the genome of a virus (see FIG. 8 ) wherein virulence mechanisms are located and thereby increased virulence may be identified and predicted. Because the inventors have provided a method of focusing on particular units of a viral genome, the skilled artisan will understand the importance of looking for Replikin sequences in any portion of the life cycle or infectious pathway of a virus.
  • predictive and virulence-related Replikin sequences may be identified in reservoirs of influenza virus such as in WSSV and TSV.
  • predictive and virulence-related Replikin sequences or Replikin Peak Genes may be identified in vectors of the influenza virus.
  • Replikin sequences or Replikin Peak Genes may be identified in hosts of the influenza virus or in any other place where viral genes may be located or wherein viral genetic information may encounter genetic information of other strains of virus, of other virus species, of vectors of the virus, of hosts of the virus, or of food sources of hosts of the virus.
  • This method of locating relevant Replikin sequences finds support in the inventors' disclosure of data providing insight into what sequence changes in influenza since 1917 were due to “drift” or “shift” or, on the other hand, what sequence changes in influenza since 1917 were due to conservation, storage in “reservoirs,” and reappearance in both intermediate vectors and eventual animal and human hosts.
  • an automated prediction of an outbreak of influenza virus is made by (1) measuring the Replikin concentration in a WSSV or TSV isolate structure, or other reservoir virus structure (2) comparing the measured concentration to the Replikin concentration determined at a previous time point in the same virus structure, and (3) observing an increase in Replikin concentration in that virus structure. If an increase has occurred, an outbreak within about one to about three years following the latest time point is predicted. In a further embodiment, the increase from one time point to another time point is statistically significant.
  • the magnitude of a Replikin increase in a virus may be a quantitative component of predicting an outbreak. For example, in H5N1 influenza virus, a two-fold (200%) increase in Replikin concentration predicted an outbreak of the virus in humans. See FIG. 16 and Example 9. Further, an eight-fold (800%) increase in the Replikin Peak Gene of the virus predicted the same outbreak, which included new morbidity and increased mortality in infections from the virus. See FIG. 17 and Example 9.
  • WSSV White Spot Syndrome Virus
  • TSV Taura Syndrome Virus
  • the concentration of Replikins in WSSV, TSV, and in influenza has been shown to be related to rapid replication and epidemics in each of these viruses.
  • An increase in the concentration of Replikin sequences has been correlated with the last three influenza pandemics of 1918, 1957 and 1968. See FIG. 11 .
  • Quantitative determination of the concentration of virus Replikins has made it possible to predict in advance recent influenza outbreaks including outbreaks of the H5N1 strain of influenza.
  • Quantitative determination of the concentration of Replikins in viruses for which nucleic acid or protein sequences are available may now be automated using the proprietary FluForecast®, which is available from Replikins, LLC, Boston Mass.
  • the H5N1 virus recently has been responsible for huge poultry losses in many countries and for several hundred human cases with approximately 50% mortality. While migratory waterfowl are known to transport H5N1 influenza virus globally, no reservoirs for the virus had yet been identified. The inventors, however, have now identified shrimp as a reservoir for homologous Replikin sequences identified in White Spot Syndrome Virus (WSSV) and Taura Syndrome Virus (TSV). The homologous Replikin sequences have been identified in both shrimp and influenza virus and in particular in the H5N1 “bird flu” strain of influenza virus in both birds and humans.
  • WSSV White Spot Syndrome Virus
  • TSV Taura Syndrome Virus
  • the inventors identified a correlation between an increase in Replikin concentration in WSSV and/or TSV and an increase in virulence (and a concomitant increase in Replikin concentration) in the H5N1 strain of influenza virus.
  • the inventors further identified homologous Replikin sequences shared within and between the virus strains including Replikin Scaffold sequences, the presence of which have been associated with high virulence, epidemics and pandemics.
  • Prediction of an outbreak of influenza may further comprise:
  • a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues and in a preferred embodiment about 28 to about 30 amino acid residues.
  • a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a more preferred embodiment about 29 to about 31 amino acid residues.
  • a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a more preferred embodiment about 29 to about 33 amino acid residues.
  • the mortality rate of shrimp for the WSSV shrimp virus is 80-100% and, as shown above, the Replikin concentration during outbreaks has exceeded 100 Replikin sequences per 100 amino acid residues.
  • the Replikin concentrations and the mortality rates in H5N1 have not been as high in ducks and chickens.
  • Replikin concentrations of twenty in the Replikin Peak Gene of H5N1 have been observed in humans in Indonesia along with mortality rates of approximately 80% in a small number of humans in 2006-2007. See U.S. application. Ser. No. 11/755,597, filed May 30, 2007, pages 36-39.
  • this evidence may reflect migration or vectoring of Replikin sequences between species, or may reflect successive independent stimulation of influenza strains in different hosts, or both, or even some other yet-to-be-determined mechanism.
  • Data that supports migration of Replikin sequences between different strains of influenza virus rather than independent synthesis has been reported in previous patent applications by the Applicants (see, e.g., U.S. Pat. No. 7,189,800 and U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006), but the present evidence of relation of shrimp viruses to influenza viruses is the first evidence of inter-virus species migration of Replikins.
  • the “infectious unit” of a virus may not be the whole virus but, instead, a relatively small constituent of the virus structure, namely Replikin sequences.
  • Replikin Scaffold sequences so far identified in WSSV begin with KK and end with H.
  • the length of the Scaffold Sequence is about 27 to 31, which is similar to the about 28 to about 29 amino acid Replikin Scaffolds generally observed in the H5N1, H1N1, H2N2, H2N3, and H7N7 strains of influenza virus.
  • the following Replikin Scaffold repeats were identified in Accession No. AAL89390, which discloses a 2000 isolate of WSSV.
  • K 66 KNVKSAKQLPHLKV H LDV K SAKQLPHLKVH 96 (SEQ ID NO: 2) K 160 KNVKSAKQLPHLKV H LDV K GVKQLLH 186 (SEQ ID NO: 3) K 239 KNVKSAKQLPHLKV L LDV R GAKQLPH 265 (SEQ ID NO: 3) K 303 KNVKSAKQLPHLKV L LDV R GAKQLPH 329 (SEQ ID NO: 4) K 397 KNVKSAKQLPHLKV L LDV R GAKQLPHLKVH 427 .
  • one aspect of the invention provides a method of predicting an influenza outbreak by correlating an increase in Replikin concentration in shrimp WSSV and/or TSV over Replikin concentration in previous isolates of WSSV and/or TSV with an increase in virulence and/or replication of influenza viruses.
  • Table 1 illustrates the history, by year or smaller time period, of the existence in the protein structure of the Goose Replikin and its homologues in other influenza Replikins. Table 1 further illustrates the history of amino acid substitutions in those homologues and the conservation of certain amino acids of the Replikin structure that are essential to the definition of a Replikin and the function of rapid replication supplied by Replikins.
  • the inventors have further established a relationship between virulent influenza virus and WSSV and TSV in the Replikin Scaffold portions of the viruses as may be seen in Table 2 below. Although there is extensive substitution, several short Replikins of theshrimp White Spot Syndrome Virus demonstrates significant homologies to the influenza virus Replikin sequences, especially with regard to length and key lysine (k) and histidine (h) residues. Similar, but less extensive, homologies are seen in Taura Syndrome Virus. These homologies suggest that the sequences are derived from a shared reservoir and/or that similar mechanisms of Replikin production are used in both virus groups.
  • a related example of virus reservoir activity in which the Replikin concentration was increased prior to an outbreak was observed in corona viruses as a group.
  • the Replikin concentration of the corona virus group increased markedly in 2002 before the outbreak of one of its members, SARS, in 2003. See FIG. 12 .
  • White Spot Syndrome Virus can now have its Replikins examined as earlier forms of virus Replikins, or as parallel morphological branches, that in either case may act as reservoirs for bird and animal Replikins such as those in influenza viruses.
  • the diagnostic and preventive uses of these Replikin findings in shrimp follow as they do in influenza and for other organisms containing Replikins.
  • falciparum is a trypanosome, both spend an essential part of their reproductive cycles in red blood cells, an unusual host cell whether in shrimp (White Spot Syndrome Virus) or man (malaria), both are fulminating, rapidly-replicating diseases with high mortality rates of their hosts, and both appear to use the same methods of increasing their high Replikin concentrations to such record highs, namely, Replikin repeats and Replikin overlap.
  • Table 3 illustrates intramolecular Replikin repeats and Replikin overlap in shrimp White Spot Syndrome Virus (WSSV) nucleocapsid protein (VP35) gene with a Replikin concentration (number of Replikins per 100 amino acids) of 103.8 (497 total Replikins per 479 amino acids).
  • the nucleocapsid protein reportedly possesses thymidine kinase and thymidylate kinase activity.
  • Replikin concentration was determined for the ribonucleotide reductase gene in WSSV from Accession No. AAL89390.
  • Accession No. AAL89390 discloses the amino acid sequence of ribonucleotide reductase translated from the total genome of a year 2000 isolate of White Spot Syndrome Virus made publicly available at Accession No. NC 003225.1.
  • the Replikin concentration as discussed above, was an unusually high at 103.8. Additionally, the Replikin concentration of the Replikin Peak Gene of the protein was even higher at 110.7.
  • the Replikin concentration of the Replikin Peak Gene was determined by dividing the number of Replikin sequences identified in the segment of the protein containing the highest concentration of Replikin sequences, 497 Replikin sequences, by the total amino acid length of the Replikin Peak Gene, 449 amino acids, to arrive at 110.7 Replikin sequences per 100 amino acids. See Example 1.
  • the amino acid sequence of the protein publicly available at Accession No. AAL89390 is of particular interest because it demonstrates an overlapping of Replikin sequences that result in very high Replikin concentrations.
  • the very large number of Replikin sequences present in the genome of the WSSV serves as a reservoir or vector for individual Replikins that are transferred or, via some other mechanism, otherwise appear in virulent strains of the influenza virus. This transfer or appearance of homologous Replikin sequences is seen in the Replikin Scaffold of the influenza virus and the Replikin Scaffold of the Shrimp White Spot Virus.
  • the amino acid sequence disclosed at Accession No. AAL89390 was further observed to contain significant Replikin Scaffold sequences.
  • the presence of Replikin Scaffold sequences in Accession No. AAL89390 was not unexpected since Replikin Scaffolds are frequently present in viral genomes when the Replikin concentration is observed to move above 3 to 4 Replikin sequences per 100 amino acids.
  • the presence of a Replikin Scaffold and a Replikin concentration above 3 or 4 correlates with viral outbreaks or epidemics and is another clear association with the rapid replication states of the isolated virus.
  • Replikin concentration has been correlated with virulence in each influenza pandemic of the 20 th century. See FIG. 11 . This shared correlation between Replikin concentration and outbreaks and mortality in WSSV and TSV and influenza provides evidence that the Replikin structure is transferable or repeatable between these viruses.
  • FIG. 11 demonstrates that frequently a one to three year stepwise increase is observed before Replikin concentration reaches a peak. This stepwise increase precedes the occurrence of an epidemic, which occurs concurrently with the Replikin peak.
  • the stepwise increase in concentration of a particular strain is a signal that a particular strain is the most likely candidate to cause an epidemic or pandemic.
  • FIG. 4 illustrates a correlation between each outbreak of H5N1 between 1997 and 2004.
  • the x-axis in FIG. 4 indicates the year and the y-axis indicates the Replikin concentration (number of Replikins per 100 amino acids).
  • the time periods for each epidemic of H5N1 are denoted in FIG. 4 by E 1 , E 2 and E 3 .
  • H5N1 bird flu
  • Replikin concentration changes in H5N1 have suggested that H5N1 virulence is continuing to increase.
  • a 2006 report nevertheless suggested that in the first probable person-to-person transmission of H5N1, “sequencing of the viral genes identified no change in the receptor-binding site of hemagglutinin or other key features of the virus. The sequences of all eight viral gene segments clustered closely with other H5N1 sequences from recent avian isolates in Thailand.” Phylogenetic analysis suggested that from the absence of evidence of “reassortment with human influenza viruses” that H5N1 is not a new variant.
  • a Replikin Peak Gene may be identified, isolated or synthesized for diagnosis, prevention or treatment of, for example, an outbreak of WSSV or an outbreak of a virus for which WSSV is a reservoir, such as influenza virus, by the method comprising: (1) obtaining a plurality of isolates of WSSV; (2) analyzing the protein sequences or protein sequence fragments of each isolate of the plurality of isolates for the presence and concentration of Replikin sequences; (3) identifying the protein sequence or the protein sequence fragment having the highest concentration of Replikin sequences in each isolate; (4) comparing the protein or protein fragment having the highest concentration of Replikin sequences in each isolate to the protein or protein fragment having the highest concentration of Replikin sequences in each of the other isolates of the plurality of isolates; (5) selecting the protein or protein fragment having the highest concentration of Replikin sequences; (6) identifying the amino acid sequence of the selected protein or protein fragment as the Replikin Peak Gene
  • a Replikin Peak Gene is a segment of the genome or a protein or segment of a protein in which the expressed gene or expressed gene segment has the highest or a higher concentration of Replikins (number of Replikins per 100 amino acids) when compared to other segments or named genes of the genome.
  • the gene or gene segment is known as the Replikin Peak Gene or Replikin Peak Gene Area.
  • a Replikin Peak Gene has been identified in H5N1 influenza virus and an increase in concentration of Replikins in the Replikin Peak Gene of H5N1 has been correlated with epidemics, increased virulence, morbidity and human mortality. See FIGS. 11-13 and U.S. Provisional Appln. Ser. No. 60/898,097.
  • a Replikin Peak Gene has been identified in the VP1 protein of Foot and Mouth Disease Virus and has been correlated with outbreaks of the virus. See U.S. Provisional Appln. Ser. No. 60/954,743 (see FIG. 3 and Examples 1-4 therein). Further, a second Replikin Peak Gene has been identified in a fragment of the VP1 protein of Foot and Mouth Disease Virus and two particular Replikin sequences within the Replikin Peak Gene Area of the virus have been correlated with virulence of Foot and Mouth Disease Virus. See id. A Replikin Peak Gene has likewise been identified in West Nile Virus, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus and Equine Influenza virus. See U.S.
  • Identification of Replikin sequences as infectious units has allowed the inventors to identify isolates having higher virulence relative to other isolates and to focus attention on the geographic area of an outbreak of virulent virus (see FIG. 10 ), the particular host of hosts of an outbreak of virulent virus (see FIG. 9 ) and the part of the genome of a virus (see FIG. 8 ) wherein virulence mechanisms are located and wherein increased virulence may be identified and predicted.
  • Identification of Replikin Peak Genes within viral genomes has improved the focus that is available to make these identifications and predictions of both virulence and mechanisms of virulence.
  • Replikin sequences may be identified in reservoirs of influenza virus such as in WSSV and TSV.
  • predictive and virulence-related Replikin sequences or Replikin Peak Genes may be identified in vectors of the influenza virus.
  • Replikin Peak Genes may be identified in hosts of the influenza virus or any other place where viral genes may be located or wherein viral genes may encounter genes of other strains of virus, other virus species, vectors or hosts.
  • FIG. 12 shows a remarkable constancy of low coronavirus Replikin concentration between 1995 and 2001 in the spike proteins, followed by a dramatic increase in 2002, one year before the SARS epidemic appeared in 2003.
  • Replikin concentration of the spike proteins in SARS then returned to their normal pre-2003 levels (correlated with the disappearance of SARS).
  • the return to normal pre-2003 levels of Replikin concentration is thus a quantitative virus structural signal of the decline of the outbreak.
  • FIG. 13 is a graph illustrating a correlation between Replikin concentration and morbidity and mortality in U.S. populations for West Nile Virus.
  • FIG. 14 is a graph illustrating a correlation between the concentration of Replikins observed in the VP1 protein of isolates of Foot and Mouth Disease Virus between 1998 and 2006 and epidemics in the United Kingdom in 2001 and 2007.
  • FIG. 15 is a graph illustrating a correlation between Replikin concentration in the Replikin Peak Gene of Equine Influenza virus and epidemics between 1977 and 2000.
  • the Replikin Peak Gene in Equine Influenza is identified as the pB1 gene area.
  • the Replikin concentration increases in the pB1 gene area of the virus, no concomitant increase in the pA or pB2 virus is observed.
  • a peptide vaccine may include a single Replikin peptide sequence or may include a plurality of Replikin sequences observed in particular virus strains.
  • the peptide vaccine is based on Replikin sequence(s) shown to be increasing in concentration over a given time period and conserved for at least that period of time.
  • a vaccine may include a conserved Replikin peptide(s) in combination with a new Replikin peptide(s) or may be based on new Replikin peptide sequences.
  • Replikin peptides can be synthesized by any method, including chemical synthesis or recombinant gene technology, and may include non-Replikin sequences, although vaccines based on peptides containing only Replikin sequences are preferred.
  • vaccine compositions of the invention also contain a pharmaceutically acceptable carrier and/or adjuvant.
  • Vaccines can be administered alone or in combination with antiviral drugs, such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
  • antiviral drugs such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
  • Vaccines may be administered to any animal capable of producing antibodies in an immune response.
  • a vaccine may be administered to a rabbit, a chicken, a pig, or a human. Because of the universal nature of Replikin sequences, a vaccine of the invention may be directed at a variety of strains of virus or a particular strain of virus.
  • isolated Replikin peptides may be used to generate antibodies, which may be used, for example to provide passive immunity in an individual.
  • Various procedures known in the art may be used for the production of antibodies to Replikin sequences.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by a Fab expression library.
  • Antibodies that are linked to a cytotoxic agent may also be generated.
  • Antibodies may also be administered in combination with an antiviral agent.
  • combinations of antibodies to different Replikins may be administered as an antibody cocktail.
  • Monoclonal antibodies to Replikins may be prepared by using any technique that provides for the production of antibody molecules. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of chimeric antibodies (Morrison et al., 1984, Proc. Nat. Acad. Sci USA, 81:6851-6855) or other techniques may be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce Replikin-specific single chain antibodies.
  • Antibodies to any peptides observed to be present in an emerging or re-emerging strain of virus and combinations of such antibodies are useful in the treatment and/or prevention of viral infection.
  • Antibody fragments that contain binding sites for a Replikin may be generated by known techniques.
  • fragments include but are not limited to F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecules and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab′)2 fragments.
  • Fab expression libraries can be generated (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • immune serum containing antibodies to one or more Replikins obtained from an individual exposed to one or more Replikins may be used to induce passive immunity in another individual or animal.
  • Immune serum may be administered via i.v. to a subject in need of treatment.
  • Passive immunity also can be achieved by injecting a recipient with preformed antibodies to one or more Replikins.
  • Passive immunization may be used to provide immediate protection to individuals who have been exposed to an infectious organism.
  • Administration of immune serum or preformed antibodies is routine and the skilled practitioner can readily ascertain the amount of serum or antibodies needed to achieve the desired effect.
  • Replikin peptides may be administered to a subject, preferably by i.v. or intramuscular injection, in order to stimulate the immune system of the subject to produce antibodies to the peptide.
  • the dosage of peptides is in the range of from about 0.1 ⁇ g to about 10 mg.
  • the dosage of pepetides is about 10 ⁇ g to about 1 mg.
  • the dosage of peptides is about 50 ⁇ g to about 500 ⁇ g. The skilled practitioner can readily determine the dosage and number of dosages needed to produce an effective immune response.
  • Replikin concentration was determined for ribonucleotide reductase publicly available at Accession No. AAL89390.
  • the amino acid sequence was translated from the total genome of a year 2000 isolate of White Spot Syndrome Virus made publicly available at Accession No. NC 003225.1.
  • the Replikin concentration in the protein was an unusually high 103.8 and the Replikin concentration of the Replikin Peak Gene of the protein was a yet higher 110.7.
  • the Replikin concentration of the protein was determined by dividing the number of Replikin sequences identified in the amino acid sequence of the protein, 497 Replikin sequences, by the total amino acid length of the protein, 479 amino acids, to arrive at 103.8 Replikin sequences per 100 amino acids.
  • the Replikin concentration of the Replikin Peak Gene was determined by dividing the number of Replikin sequences identified in the segment of the protein containing the highest concentration of Replikin sequences, 497 Replikin sequences, by the total amino acid length of the Replikin Peak Gene, 449 amino acids, to arrive at 110.7 Replikin sequences per 100 amino acids.
  • the amino acid sequence of the protein publicly available at Accession No. AAL89390 is of particular interest because it demonstrates an overlapping of Replikin sequences that results in very high Replikin concentrations, comparable to P. falciparum .
  • the high concentrations of Replikin sequences provide a reservoir for transfer to influenza viruses.
  • a Replikin Peak Gene is the segment of a protein having the highest concentration of continuous, uninterrupted, overlapping, Replikin sequences. In the sequence disclosed below, the Replikin Peak Gene is underlined. The Replikin Peak Gene is observed to occupy most of the disclosed protein. The expansiveness of the Replikin Peak Gene across most of the amino acid sequence of the protein is highly unusual and creates the remarkably high Replikin concentration. The amino acid sequence further contains significant Replikin Scaffold sequences. The following Replikin Scaffold repeats were identified in Accession No. AAL89390.
  • K 66 KNVKSAKQLPHLKV H LDV K SAKQLPHLKVH 96 (SEQ ID NO: 2) K 160 KNVKSAKQLPHLKV H LDV K GVKQLLH 186 (SEQ ID NO: 3) K 239 KNVKSAKQLPHLKV L LDV R GAKQLPH 265 (SEQ ID NO: 3) K 303 KNVKSAKQLPHLKV L LDV R GAKQLPH 329 (SEQ ID NO: 4) K 397 KNVKSAKQLPHLKV L LDV R GAKQLPHLKVH 427
  • Replikin concentration was determined for a functionally undefined protein in the genome of an isolate of White Spot Syndrome Virus from 2000 made publicly available at NP 478030.
  • the Replikin Concentration in the protein was again an unusually high 97.6 Replikin sequences per 100 amino acids.
  • the Replikin concentration was determined by dividing the number of Replikin sequences identified in the amino acid sequence of the protein, 361 Replikin sequences, by the total amino acid length of the protein, 370 amino acids.
  • NP 478030 The amino acid sequence of NP 478030 is of interest because, like the protien in Example 1, it demonstrates an overlapping of Replikin sequences that results in very high Replikin concentration comparable to the highly-replicating P. falciparum of malaria. These high concentrations of Replikin sequences again provide a reservoir of Replikins for transfer to influenza viruses.
  • WSSV White Spot Syndrome Virus
  • This continued decline of WSSV into “quiescent” or “dormant” levels in 2006-2007 is demonstrated in mean Replikin concentrations for viruses isolated during 2005-2007 that are very low as compared to years wherein the virus demonstrated greater virulence, such as 2001. See Table 4 in Example 4 below.
  • the continued quiescence in WSSV in 2007 may be contrasted with an observed rising of Replikin concentration in Taura Syndrome Virus Replikin during this period.
  • ABS00973 contains a single Replikin sequence in the entire disclosed amino acid sequence of 240 residues. The single Replikin sequence is underlined. The Replikin concentration of Accession No. ABS00973 is an inordinately low 0.5.
  • Replikin concentrations were determined for all amino acid sequences for WSSV with accession numbers publicly available at www.pubmed.com.
  • the amino acid sequences were scanned for Replikin sequences of 7 to 50 amino acids comprising (1) at least one lysine residue located at a first terminus of the sequence and at least one lysine residue or at least one histidine residue located at a second terminus of the sequence; (2) a first lysine residue located six to ten residues from a second lysine residue; (3) at least one histidine residue; and (4) at least 6% lysine residues.
  • the total number of Replikin sequences was determined for each available accession number.
  • the Taura Syndrome Shrimp Virus is less virulent than WSSV and the structure of the TSV Replikin Scaffold is less closely related to influenza virus than are the structures of WSSV Replikin Scaffolds.
  • TSV had a Replikin concentration of 3.3.
  • TSV had a lower mean Replikin concentration, as low as 0.7, and its Replikin Scaffold disappeared.
  • the Replikin Scaffold reappeared, with an increase in lysines and histidines, and a commensurate increase in Replikin concentration to 3.9, followed by an increase in TSV outbreaks in 2006-2007.
  • PubMed Code AAM73766 Description: Isolated: 2001 Source: Taura syndrome virus M 1 P 2 A 3 N 4 P 5 V 6 E 7 I 8 D 9 N 10 F 11 D 12 T 13 T 14 T 15 S 16 G 17 G 18 L 19 I 20 P 21 G 22 G 23 S 24 V 25 T 26 N 27 S 28 E 29 G 30 S 31 T 32 I 33 L 34 M 35 N 36 D 37 I 38 P 39 I 40 T 41 N 42 Q 43 N 44 V 45 V 46 L 47 S 48 K 49 N 50 V 51 T 52 D 53 N 54 L 55 F 56 E 57 V 58 Q 59 D 60 Q 61 A 62 L 63 I 64 E 65 S 66 L 67 S 68 R 69 D 70 V 71 L 72 L 73 H 74 N 75 D 76 S 77 W 78 T 79 S 80 S 81 D 82 D 83 E 84 I 85 G 86 T 87 T 88 M 89 T 90 Q 91 E 92 Q 93 L 94 A 95 T 96 E 97 F 98 N 99 Q 100 P 101
  • Replikin concentrations were determined for all amino acid sequences for Taura Syndrome Virus with accession numbers publicly available at www.pubmed.com.
  • the amino acid sequences were scanned for Replikin sequences of 7 to 50 amino acids comprising (1) at least one lysine residue located at a first terminus of the sequence and at least one lysine residue or at least one histidine residue located at a second terminus of the sequence; (2) a first lysine residue located six to ten residues from a second lysine residue; (3) at least one histidine residue; and (4) at least 6% lysine residues.
  • the total number of Replikin sequences was determined for each available accession number.
  • the virulence of the four TSV isolates was compared in an independent laboratory, without any knowledge of the Replikin concentrations.
  • the virulence was compared through a per os laboratory infection in juvenile Litopenaeus vannamei (Kona stock, Oceanic Institute, Hawaii). The results showed that the Caribbean isolate is the most virulent, the Thailand isolate is the second most virulent, followed by the Hawaii isolate, and the Venezuela isolate, which is the least virulent.
  • Severe (G4) TSV infection was detected in 2 out of 3 shrimp, and another shrimp showed a moderate to high grade (G3) of infection. Lymphoid organ spheroids were found at severities of G2 and G3.
  • G2 Moderate level of TSV infection was detected in 2 shrimp collected at day 4. Lymphoid organ spheroids were found at severities of G3 and G4.
  • TSV TSV isolate Mean TSV copies/:l RNA Caribbean 2.7 ⁇ 10 6 (4.8 ⁇ 10 5 -4.4 ⁇ 10 6 ) Thailand 2.7 ⁇ 10 6 (4.3 ⁇ 10 5 -7.5 ⁇ 10 6 ) Hawaii 5.2 ⁇ 10 7 (2.3 ⁇ 10 7 -7.5 ⁇ 10 7 ) Venezuela 6.5 ⁇ 10 5 (6.5 ⁇ 10 2 -2.0 ⁇ 10 5 )
  • the phenomenon of “resistance” to infection appears to be based in a “primitive immune system” perhaps similar to the “toll receptor” and related systems.
  • the term “increased resistance” is used for the observed phenomenon and Replikin feed is used rather than “vaccine” for the administered substance which increases resistance.
  • the surviving shrimp of the first challenge were then set up in a fresh culture, fed for an additional two weeks with feed containing Replikin sequences, then again challenged with the Hawaii strain of Taura syndrome virus.
  • the Replikin sequence supplemented feed was maintained while the survivors were again challenged repeatedly by the same virus, in repeated cycles, until 100% of the shrimp survived the TSV challenge.
  • the inventors analyzed and compared percent human mortality from H5N1 infections in years 2005 through the first quarter of 2007 to mean concentration of Replikin sequences in (1) the whole genome (2) the polymerase gene, (3) the pB1 gene area, (4) the pB2 gene area, and (5) the pA gene area, respectively, of H5N1 influenza strains isolated in 2003 through the first quarter of 2007.
  • the following data were observed:
  • Table 10 provides mortality data for H5N1 infections from 2005 through 2007 and does not include earlier mortality data.
  • Mortality data prior to 2005 has not been included in Table 10 (or in the data in FIGS. 1-3 ) because data prior to 2005 is inconsistent and understood by those of skill in the art to contain errors, including errors caused by under-reporting.
  • the usual figures cited for 1997 are: 30 human cases, 8 deaths with mortality rate of about 27%. The number of cases (morbidity) and the number who died (mortality) that were not reported is unknown, but suspected to be significant.
  • Table 10 provides strong confirmation of the power and validity of the methodology of predicting changes in virulence and outbreaks of virus with changes in Replikin concentration.
  • These data represent an objective test of the method of independently selecting and examining several thousand individual accession numbers within approximately 12 million total accession numbers in PubMed wherein each selection is independently submitted to the PubMed data base under a separate request using objective software. If there were not a reliable principle and a reliable method underlying each request, the potential for obtaining random results, or no results, or results which do not track each other at p ⁇ 0.001 would markedly increase.
  • Table 10 provides results wherein p was less than 0.001 between each group as compared one to another.
  • Table 10 the structures that are correlated have, to the knowledge of the inventors, not been correlated before, that is, the inventors have examined the relationship of one internal virus structure to another internal virus structure or structures (e.g., three-way relationship between whole virus gene area, polymerase, and Replikin Peak Gene area) and have examined the external relation of these two or more internal structures to a host result of the virus infection, that is, percent mortality.
  • one internal virus structure to another internal virus structure or structures (e.g., three-way relationship between whole virus gene area, polymerase, and Replikin Peak Gene area) and have examined the external relation of these two or more internal structures to a host result of the virus infection, that is, percent mortality.
  • Table 10 represents consistent reproducible data, on repeated trials, which is the essence of the reliability of any method.
  • Table 10 provides independent data on (1) whole virus concentration of Replikins, (2) only the polymerase concentration of Replikins, and (3) only the Replikin Peak Gene concentration of Replikins.
  • the data is then correlated with H5N1 mortality three times, namely in 2005, 2006 and 2007.
  • the absence of significant changes in the pA and pB2 gene areas provides a control.
  • the method measures Replikin concentration three ways, each of which correctly predict mortality, independently, thereby confirming the method, and further illustrating in the process, the magnifying function of the Replikin Peak Gene.

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Abstract

The present invention provides a method of predicting outbreaks in influenza virus comprising monitoring concentrations of small peptides having defined structure in influenza viruses, and in particular viral pathogens of shrimp.

Description

This application claims priority to U.S. Provisional Appln. Ser. No. 60/935,816, filed Aug. 31, 2007, U.S. Provisional Appln. Ser. No. 60/935,499 filed Aug. 16, 2007, U.S. Provisional Appln. Ser. No. 60/954,743, filed Aug. 8, 2007, U.S. Provisional Appln. Ser. No. 60/898,097, filed Jan. 30, 2007, U.S. Provisional Appln. Ser. No. 60/880,966, filed Jan. 18, 2007, and U.S. Provisional Appln. Ser. No. 60/853,744, filed Oct. 24, 2006, each of which is incorporated herein by reference in its entirety. This application additionally incorporates herein by reference: U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006, U.S. application Ser. No. 11/755,597, filed May 30, 2007, U.S. application Ser. No. 11/116,203, filed Apr. 28, 2005, U.S. application Ser. No. 10/860,050, filed Jun. 4, 2004, U.S. application Ser. No. 10/189,437, filed Jul. 8, 2002, U.S. application Ser. No. 10/105,232, filed Mar. 26, 2002, now U.S. Pat. No. 7,189,800, U.S. application Ser. No. 09/984,057, filed Oct. 26, 2001, and U.S. application Ser. No. 09/984,056, filed Oct. 26, 2001, now U.S. Pat. No. 7,176,275, each in its entirety.
TECHNICAL FIELD OF THE INVENTION
This invention relates generally to predicting influenza outbreaks through monitoring quantitative changes in the concentration of a class of peptide sequences known as Replikins in virus proteins, protein fragments, or genomes.
BACKGROUND OF THE INVENTION
Rapid replication is characteristic of virulence in, among other things, certain bacteria, viruses and malignancies. The inventors have described a quantitative chemistry common to rapid replication in different viruses and organisms. The chemistry of rapid replication described by the inventors is present in a family of conserved small protein sequences related to rapid replication, Replikins. An increase in the concentration of these Replikin sequences encoded in the genome of different strains of influenza virus has been correlated with an increase in the virulence of influenza. A correlation between increased concentrations of Replikin sequences and increased replication and virulence has likewise been observed in a range of viruses and organisms. Replikin sequences offer new targets for developing effective methods of predicting and treating influenza outbreaks. There continues to exist a particular need in the art for methods of predicting viral outbreaks.
Influenza is an acute respiratory illness of global importance. Despite international attempts to control influenza virus outbreaks through vaccination, influenza infections remain an important cause of morbidity and mortality. Worldwide influenza pandemics have occurred at irregular and previously unpredictable intervals throughout history and it is expected that influenza pandemics will continue to occur in the future. The impact of pandemic influenza is substantial in terms of morbidity, mortality and economic cost.
Influenza vaccines remain the most effective defense against influenza virus, but because of the ability of the virus to mutate, and the availability of non-human host reservoirs, it is expected that influenza will remain an emergent or re-emergent infection. Global influenza surveillance indicates that influenza viruses may vary within a country and between countries and continents during an influenza season. Virologic surveillance is of importance in monitoring antigenic shift and drift. Disease surveillance is also important in assessing the impact of epidemics. Both types of information have provided the basis of vaccine composition and use of antivirals. However, there has traditionally been only annual post hoc hematological classification of the increasing number of emerging influenza virus strains, and no specific chemical structure of the viruses was traditionally identified as an indicator of approaching influenza epidemic or pandemic. Until recently, the only basis for annual classification of influenza virus as active, inactive or prevalent in a given year was the activities of the virus hemagglutinin and neuraminidase proteins.
The small peptide structure called Replikins has now been identified within influenza virus proteins and correlated with an increase in virulence. A Replikin sequence is an amino acid sequence of 7 to about 50 amino acids comprising a Replikin motif. A Replikin motif comprises (1) at least one lysine residue located at a first terminus of the motif and at least one lysine residue or at least one histidine residue located at a second terminus of the motif, (2) a first lysine residue located six to ten residues from a second lysine residue; (3) at least one histidine residue; and (4) at least 6% lysine residues. A Replikin sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine. A Replikin peptide or Replikin protein is a peptide or protein consisting of a Replikin sequence.
Higher concentrations of Replikin sequences in the genomic code are associated with a variety of infectious agents including HIV, plant viruses, and a range of pathogenic animal and human viruses including flu viruses. Further, the correlation between the concentration of Replikin sequences in viral or organismal proteins and major outbreaks of disease is significant. Replikin sequences generally have been found to be conserved in both intrastrain and interstrain influenza viruses for as long as 89 years based on data going back to the 1917-18 flu pandemic. Concentration of Replikin sequences in viral genomes has been shown to increase prior to strain-specific flu outbreaks.
Within the last century there have been three influenza pandemics, each strain specific: H1N1 in 1918; H2N2 in 1957; and H3N2 in 1968. The inventors have established that prior to each pandemic there was a strain-specific increase in the concentration of Replikin sequences within the strain. The strain-specific increase in Replikin concentration was followed by a decrease in Replikin concentration and several years later a rebound increase in Replikin concentration associated with a strain-specific rebound epidemic. The Replikin algorithm provided the first chemistry that correlated with influenza epidemics and pandemics.
A similar correlation between the outbreaks of H5NI (Bird Flu) between 1997 and 2007 and the concentration of Replikin sequences in the viral proteins during each of those years has been demonstrated. Likewise, a correlation has been established between the global outbreak of SARS coronavirus in 2003 and an increase in the concentration of Replikin sequences in the proteins of coronavirus. In another study, Replikins in two strains of human HIV-1 virus demonstrated that the Replikin concentration in the rapidly replicating strain was six fold greater than that of a slowly replicating strain. No instances of rapid replication have been observed in all the viruses and organisms examined wherein the Replikin concentration did not significantly increase as compared to the Replikin concentration in the dormant state.
The highest concentration of Replikin sequences in an organism or virus that has to date been analyzed and reported is 111 Replikin sequences per 100 amino acids in the extraordinarily-rapidly-replicating parasitic protozoa Plasmodium falciparum (reportedly responsible for 90% of malarial deaths in humans) (herein sometimes referred to as malaria). P. falciparum has been observed to replicate 11,000 times in 48 hours during passage of the parasite from liver to blood in the host.
It has been believed that changes in the activity of different influenza strains are related to random sequence changes in influenza hemagglutinins, which in turn are the products of substitutions effected by one of two poorly understood processes: i) antigenic drift, thought to be due to the accumulation of a series of point mutations in the hemagglutinin molecule, or ii) antigenic shift, in which the changes are so great that genetic reassortment is postulated to occur between the viruses of human and non-human hosts. The data provided by the inventors suggests that change in activity in different influenza strains, rather than being related to non-specific random sequence changes, is based upon, or related to, an increase in concentration of strain-specific Replikins. Data were also examined for insight into which sequence changes were due to “drift” or “shift” and which were due to conservation, storage in “reservoirs,” and reappearance. The data has shown that the epidemic-related increase in Replikin concentration is not due to the duplication of existing Replikins in the hemagglutinin of the emerging strain, but, instead is due to the reappearance of at least one Replikin composition from 1 to up to 59 years after its disappearance, plus (in the A strains only) the emergence of new strain-specific Replikin compositions. See U.S. Pat. No. 7,189,800 issued Mar. 13, 2007 (Tables 3-6).
In monitoring Replikin sequences in influenza virus, the inventors have additionally identified a sub-family of conserved Replikin sequences known as Replikin Scaffolds or Replikin Scaffold sequences. Replikin Scaffolds were initially identified in conserved structures in particularly virulent influenza viruses. Included among these strains were the viruses causing the pandemics of 1918, 1957, 1968 and virulent strains of the H5N1 “bird flu” strain of influenza virus. Analogues of Replikin Scaffold sequences have since been identified in the virulent and rapidly replicating SARS coronavirus. See U.S. Published Application No. 2007/0026009.
Scaffolding of Replikin sequences homologous but not identical to the algorithm of the identified Replikin Scaffold has also been identified in P. falciparum. Replikin scaffolding in general has been related to an increase in Replikin concentrations in pathogenic genomes where it has been identified. In P. falciparum, scaffolding contributes significantly to the very high Replikin concentration noted in the proteins of the protozoa.
There is a need in the art for methods of predicting increases in virulence of influenza prior to outbreaks. There is likewise a need in the art for methods of preventing and treating outbreaks caused by virulent strains of influenza. Because of the annual administration of influenza vaccines and the short period of time when a vaccine can be administered, strategies directed at improving vaccine coverage are of critical importance.
SUMMARY OF THE INVENTION
The present invention provides a method of determining an increased probability of an outbreak of influenza virus within about one to about three years following an increase in Replikin concentration in an isolate of White Spot Syndrome Virus or an isolate of Taura Syndrome Virus comprising identifying said increase in the concentration of Replikin sequences in at least one first isolate of White Spot Syndrome Virus or Taura Syndrome Virus as compared to at least one other isolate of White Spot Syndrome Virus or Taura Syndrome Virus wherein said at least one first isolate is isolated at least six months later than said at least one other isolate is isolated and wherein when said at least one first isolate is a White Spot Syndrome Virus isolate said at least one other isolate is a White Spot Syndrome Virus isolate and when said at least one first isolate is a Taura Syndrome Virus isolate said at least one other isolate is a Taura Syndrome Virus isolate, and wherein said increase in the concentration of Replikin sequences signifies the increased probability of the outbreak of influenza virus within about one to about three years following said increase in the concentration of Replikin sequences.
In an embodiment of the method of determining an increased probability of an outbreak of influenza virus, said identification of an increase in Replikin concentration in said at least one first isolate comprises (1) determining the concentration of Replikin sequences in (i) a plurality of isolates of White Spot Syndrome Virus wherein said at least one first isolate has been isolated about six months to about three years later than said at least one other of said isolates of White Spot Syndrome Virus, or (ii) a plurality of isolates of Taura Syndrome Virus wherein said at least one first isolate of Taura Syndrome Virus is isolated about six months to about three years later than at least one other isolate of Taura Syndrome Virus.
An embodiment of the present invention provides a method of determining an increased probability of an outbreak of influenza virus comprising:
  • (1) obtaining a plurality of isolates of White Spot Syndrome Virus or a plurality of isolates of Taura Syndrome Virus wherein at least one of said isolates of White Spot Syndrome Virus is isolated about six months to about 3 years later than at least one other of said isolates of White Spot Syndrome Virus or wherein at least one of said isolates of Taura Syndrome Virus is isolated about six months to about 3 years later than at least one other of said isolates of Taura Syndrome Virus;
  • (2) analyzing the amino acid sequence of at least one encoded protein or protein fragment or a combination of encoded proteins and/or protein fragments in each isolate of the plurality of isolates of White Spot Syndrome Virus or the plurality of isolates of Taura Syndrome Virus for the presence and concentration of encoded Replikin sequences;
  • (3) comparing the concentrations of encoded Replikin sequences in the at least one protein or protein fragment or in the combination of proteins or protein fragments in each isolate of the plurality of isolates of White Spot Syndrome Virus or in each isolate of the plurality of isolates of Taura Syndrome Virus;
  • (4) identifying an increase in the concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus or in said plurality of isolates of Taura Syndrome Virus over at least one time period of about six months or greater; and
  • (5) predicting an outbreak of influenza within about one to about three years following said identified increase in the concentration of Replikin sequences in said isolates of White Spot Syndrome Virus or in the concentration of Replikin sequences in said isolates of Taura Syndrome Virus.
In a further embodiment of the invention, the increase in concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus or in said plurality of isolates of Taura Syndrome Virus over at least one time period of about six months or greater is an increase in the mean concentration of at least two isolates as compared to at least two other isolates isolated at a later time point of about six months or greater. In a further embodiment, the increase in concentration is an increase in the mean concentration of at least 10 isolates as compared to at least 10 other isolates. In a further embodiment, the increase in concentration is an increase in the mean concentration of all isolates available at a given time point as compared to all isolates available at another given time point, wherein said time points are separated by about six months or greater. In a further embodiment, the mean concentration of said plurality of isolates is increased over the at least one time period when the mean concentration of the isolates at a later time point is greater than the mean concentration plus one standard deviation of the earlier isolates. In a further embodiment, the mean concentration of said plurality of isolates is increased over the at least one time period when the mean concentration of the isolates at a later time point is greater than the mean concentration plus two standard deviations of the earlier isolates. In a further embodiment of the invention, analyzing the combination of encoded proteins and/or protein fragments preferably comprises all amino acid sequences available for White Spot Syndrome Virus or all amino acid sequences available for Taura Syndrome Virus at a given time point, such as, for example, all amino acid sequences available in a first year and all amino acid sequences available in some other year. In another embodiment, the combination comprises at least 100 amino acid sequences. In another embodiment, the combination comprsises at least 50 amino acid sequences. In another embodiment, the combination comprises at least 10 amino acid sequences. In another embodiment, the combination comprises at least 2 amino acid sequences.
In further embodiment of the invention, the method of predicting the outbreak of influenza virus may further comprise:
  • (1) identifying at least one Replikin Scaffold sequence present in at least one isolate of the plurality of isolates of White Spot Syndrome Virus identified as having an increase in the concentration of Replikin sequences as compared to at least one other isolate of the plurality of isolates of White Spot Syndrome Virus over a time period of about six months or greater or identifying at least one Replikin Scaffold sequence present in at least one isolate in the plurality of isolates of Taura Syndrome Virus identified as having an increase in the concentration of Replikin sequences as compared to at least one other isolate of the plurality of isolates of Taura Syndrome Virus over a time period of about six months or greater;
  • (2) identifying at least one Replikin Scaffold sequence present in at least one isolate of a strain of influenza virus, wherein said strain of influenza is isolated within the time period in which the increase in concentration of Replikin sequences is identified or wherein said strain of influenza is isolated within said time period of about one to about three years after said increase in concentration of Replikin sequences is identified; and
  • (3) predicting the strain of influenza virus in which the at least one Replikin Scaffold sequence is identified as the strain of virus that will likely cause the predicted outbreak of influenza virus, wherein a Replikin Scaffold is a peptide having about 16 to about 34 amino acids comprising (a) a terminal lysine and optionally a lysine immediately adjacent to the terminal lysine; (b) a terminal histidine and optionally a histidine immediately adjacent to the terminal histidine, (c) a lysine within about 6 to about 10 amino acids of another lysine; and (d) at least 6% lysines. In an embodiment, the likelihood of an outbreak of influenza is preferably greater than 50%, more preferably greater than 60%, and even more preferably greater than 75%.
In a further embodiment of the invention, the Replikin Scaffold is a peptide having about 27 to about 33 amino acids. In an influenza virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a preferred embodiment about 28 to about 30 amino acid residues. In a White Spot Syndrome Virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a preferred embodiment about 29 to about 31 amino acid residues. In a Taura Syndrome Virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a preferred embodiment about 29 to about 33 amino acid residues.
In an embodiment of the invention, the method of predicting an outbreak of influenza virus may comprise the prediction of an outbreak of any strain of influenza virus. In a further embodiment, the method of predicting an outbreak of influenza virus may comprise the prediction of an outbreak of influenza A. In a preferred embodiment, the outbreak of influenza A may be an outbreak of H5N1 (“bird flu”) virus.
In a further embodiment of the invention, a plurality of isolates of White Spot Syndrome Virus may be obtained and analyzed for Replikin concentration or a plurality of isolates of Taura Syndrome Virus may be obtained and analyzed for Replikin concentration, or both in order to predict an outbreak of influenza virus.
In a further embodiment of the invention, the concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus or said plurality of isolates of Taura Syndrome Virus over at least one time period of about six months or greater may increase by one or more Replikin sequences per 100 amino acids, may increase by five or more Replikin sequences per 100 amino acids, may increase by ten or more Replikin sequences per 100 amino acids, may increase by fifty or more Replikin sequences per 100 amino acids, may increase by 90 or more Replikin sequences per 100 amino acids, or may increase by 100 or more Replikin sequences per 100 amino acids.
The identified increase in concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus or said plurality of isolates of Taura Syndrome Virus may occur over any time period including over one month or more, over six months or more, over one year or more, or over three years or more. In an embodiment of the invention, the identified increase in concentration of encoded Replikin sequences occurs over at least six months to about five years. In a further embodiment of the invention, the identified increase in concentration of encoded Replikin sequences preferably occurs over at least six months to about three years.
In a further embodiment of the invention, the method of predicting an outbreak of influenza may predict an outbreak within about one month to about five years or more following the identification of an increase in Replikin concentration in a plurality of isolates of White Spot Syndrome Virus or in a plurality of isolates of Taura Syndrome Virus. In a further embodiment of the invention, the method may predict an outbreak within several months to about three years.
In another embodiment, the method may predict an outbreak within about one year to about five years. In a further embodiment of the invention, the method may predict an outbreak within several months to about one year. In another embodiment, the method may predict an outbreak within about one year to about three years.
Another aspect of the invention provides a method of predicting an outbreak of influenza virus comprising analyzing Replikin concentration in reservoirs for influenza virus. A non-limiting embodiment of the invention provides a method of predicting an outbreak of influenza virus comprising:
  • (1) obtaining a plurality of isolates of a first virus from at least one reservoir of Replikin sequences shared by influenza virus wherein the reservoir of Replikin sequences shared by influenza is any source of Replikin sequences that may be shared with an influenza virus including any host of influenza virus, any food source of a host of the influenza virus, any vector of influenza virus, or any substance wherein the genetic information of the influenza virus may be shared, mingled, mixed, exchanged or come into the proximity of the Replikin sequences of the reservoir;
  • (2) analyzing the amino acid sequence of at least one encoded protein or protein fragment or a combination of encoded proteins and/or protein fragments in each isolate of the plurality of isolates of said first virus for the presence and concentration of encoded Replikin sequences;
  • (3) comparing the concentrations of encoded Replikin sequences in the at least one protein or protein fragment or in the combination of proteins or protein fragments in each isolate of the plurality of isolates of said first virus;
  • (4) identifying an increase in the concentration of encoded Replikin sequences in said plurality of isolates of said virus over at least one time period of about six months or greater; and
  • (5) predicting an outbreak of influenza within about one to about three or more years following said identified increase in the concentration of Replikin sequences in said isolates of said first virus.
In a further non-limiting embodiment, the reservoir is shrimp. In another non-limiting embodiment, the reservoir is a bird. In another non-limiting embodiment, the reservoir is a migratory bird. In another non-limiting embodiment, the reservoir is a chicken, duck, goose, or other domestic bird.
In a further non-limiting embodiment, the method of predicting an outbreak of influenza virus further comprises:
  • (1) identifying at least one Replikin Scaffold sequence present in at least one isolate of the plurality of isolates of said first virus identified as having an increase in the concentration of Replikin sequences as compared to at least one other isolate of the plurality of isolates of said first virus over a time period of about six months;
  • (2) identifying at least one Replikin Scaffold sequence present in at least one isolate of a strain of influenza virus, wherein said strain of influenza is isolated within a time period in which the increase in concentration of Replikin sequences is identified or wherein said strain of influenza is isolated within a time period of about one year to about three years after said increase in concentration of Replikin sequences is identified; and
  • (3) predicting the strain of influenza virus in which the at least one Replikin Scaffold sequence is identified as the strain of virus that will cause the predicted outbreak of influenza virus, wherein a Replikin Scaffold is a peptide consisting of about 16 to about 34 amino acids comprising (1) a terminal lysine and optionally a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and optionally a histidine immediately adjacent to the terminal histidine, (3) a lysine within about 6 to about 10 amino acids of another lysine; and (4) at least 6% lysines.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a correlation between (1) the mean concentration and standard deviation of Replikin sequences observed in amino acid sequences of White Spot Syndrome Virus (WSSV) isolated between 1995 and 2007 that have publicly available accession numbers at www.pubmed.com, (2) the mean concentration of Replikin sequences in the pB1 gene area of H5N1 strains of influenza virus isolated in 2003, 2004, 2005, 2006, and 2007 that have publicly available accession numbers at www.pubmed.com, and (3) the mean mortality rate for humans infected with H5N1 influenza virus isolated in 2005, 2006 and 2007. The data demonstrate an exceptionally high Replikin sequence concentration in WSSV in 2000 followed within three years by an increase in Replikin sequence concentration in H5N1 isolates (from 2003 through 2007) and a concomitant outbreak of H5N1 influenza in humans with rising mortality.
FIG. 2 illustrates a correlation between (1) the mean concentration and standard deviation of Replikin sequences observed in Taura Syndrome Virus (TSV) isolated between 2000 and 2005 that have publicly available accession numbers at www.pubmed.com, (2) the mean concentration of Replikin sequences in the pB1 gene area of H5N1 strains of influenza virus isolated in 2003, 2004, 2005, 2006, and 2007 that have publicly available accession numbers at www.pubmed.com, and (3) the mean mortality rate for humans infected with H5N1 influenza virus isolated in 2005, 2006, and 2007. The data demonstrate a higher Replikin sequence concentration in TSV in 2000 followed by a marked decrease in Replikin concentration in TSV in 2001 followed within three years by an increase in Replikin sequence concentration in H5N1 isolates (from 2003 through 2007) and a concomitant outbreak of H5N1 influenza in humans with increasing mortality.
FIG. 3 illustrates a correlation between Replikin concentration in the H5N1 strain of influenza virus and human mortality from 2005 through the first quarter of 2007. FIG. 3 contains a graph comparing percent human mortality from H5N1 infections in years 2005 through the first quarter of 2007, mean concentration of Replikin sequences in the genome of H5N1 influenza strains isolated in 2003 through the first quarter of 2007 and mean concentration of Replikin sequences in the pB1 gene area of H5N1 influenza strains isolated in 2003 through the first quarter of 2007. Mean human mortality in deaths per 10 infections in a given year is represented by the black columns, mean Replikin concentration in the whole virus genome in a given year is represented by the light gray columns, mean Replikin concentration in the pB1 gene area in a given year is represented by the dark gray columns, and standard deviations are represented by the white columns.
FIG. 4 illustrates that an increasing Replikin concentration of hemagglutinin protein in the H5N1 strain of influenza virus preceded three “Bird Flu” Epidemics. In H5N1 influenza, the increasing strain-specific Replikin concentration (Replikin Count, Means+/−SD) 1995 to 1997 preceded the Hong Kong H5N1 epidemic of 1997 (E1); the increase from 1999 to 2001 preceded the epidemic of 2001 (E2); and the increase from 2002 to 2004 preceded the epidemic in 2004 (E3). The decline in 1999 occurred with the massive culling of poultry in response to the E1 epidemic in Hong Kong.
FIG. 5 illustrates a direct sequential correlation between Replikin concentration in isolates of Taura Syndrome Virus (TSV) collected from Belize, Thailand, Hawaii and Venezuela, respectively, and mean number of days until 50% mortality in Litopenaeus vannamei shrimp challenged with the respective TSV isolates on day zero. Statistical differences between the Replikin concentration for each isolate are significant at a level of p<0.001.
FIG. 6 illustrates a direct sequential correlation between Replikin concentration in isolates of Taura Syndrome Virus (TSV) collected from Belize, Thailand, Hawaii and Venezuela, respectively, and mean cumulative survival of Litopenaeus vannamei shrimp at 15 days after challenge with the respective TSV isolate. Statistical differences between the Replikin concentration for each isolate are significant at a level of p<0.001.
FIG. 7 illustrates that cumulative survival of Litopenaeus vannamei shrimp challenged with four different Taura Syndrome Virus isolates over 15 days (unless 100% mortality occurred prior to 15 days) correlates sequentially with the Replikin concentration of Open Reading Frame 1 (ORF1) of each isolate. Translated amino acid sequences of ORF1 of the genome of individual isolates of TSV from Belize, Thailand, Hawaii and Venezuela were analyzed for Replikin concentration. Replikin concentration was determined to be 3.5 for the Belize isolate, 3.4 for the Thailand isolate, 3.3 for the Hawaii isolate and 3.0 for the Venezuela isolate. Graph A illustrates observed percent survival in three trials of shrimp challenged with the Belize isolate of TSV. In one trial, total mortality was observed on day 6. In the other two trials, total mortality was observed on day 11. Graphs B, C and D illustrate observed percent survival of shrimp challenged with the Thailand isolate, the Hawaii isolate and the Venezuela isolate, respectively, each in three trials over 15 days. In the Thailand isolate, a mean of 80% percent mortality was observed on day 15. In the Hawaii isolate, a mean of 78.3% mortality was observed on day 15. In the Venezuela isolate, a mean of 58.3% mortality was observed on day 15.
FIG. 8 illustrates the localization of the pB1 gene area as the Replikin Peak Gene in the genome of the H5N1 strain of influenza virus. FIG. 8 discloses a bar graph depicting the number (with standard deviation) of Replikins per 100 amino acids in the eight genes of H5N1 influenza virus identified annually in humans between 2003 and 2006. A significant increase is observed in the pB1 gene area as compared to the other seven genes of the H5N1 influenza virus strain.
FIG. 9 illustrates a correlation between an increase in Replikin concentration in the pB1 gene area (Replikin Peak Gene) of the H5N1 strain of influenza virus in various bird species and humans over time with an increase in virulence of the H5N1 strain of influenza. FIG. 9 discloses a graph depicting the number (with standard deviation) of Replikins per 100 amino acids in the pB1 gene area (Replikin Peak Gene) of H5N1 influenza virus strains identified annually in duck and chicken in years 2001 through 2006, in goose in years 2001 and 2003 through 2006, and in human in years 2003 through 2006. The increase in Replikin concentration in these species over time correlates with an increase in virulence of the H5N1 virus strain over the same time period and correlates with an increase in mortality rate in humans during that time period. Changes in Replikin concentration in the Replikin Peak Gene of the H5N1 isolates in FIG. 9 allows for identification of those hosts in which the influenza virus strain is more virulent than other hosts.
FIG. 10 is a bar graph depicting the number (with standard deviation) of Replikins per 100 amino acids in the pB1 gene area (Replikin Peak Gene) of H5N1 influenza virus strains identified annually in humans in Japan, Russia, Egypt, China, Vietnam, Thailand and Indonesia between 2003 and 2006. Incidence of human morbidity and mortality in the Indonesian outbreak were exceptionally high and evidence of possible human to human transmission was observed. Changes in Replikin concentration in the Replikin Peak Gene of the H5N1 isolates in FIG. 10 allows for identification of those geographic areas in which the influenza virus strain is more virulent than other geographic areas.
FIG. 11 is a graph correlating Replikin Count in each of three strains of influenza virus responsible for one of three pandemics during the 20th century with virulence. Within the last century there have been three influenza pandemics, each pandemic being strain specific: H1N1 in 1918; H2N2 in 1957; and H3N2 in 1968. In the reported amino acid sequences of the strains responsible for each pandemic, there is a strain-specific increase in Replikin concentration correlated with the major pandemic within the strain, followed by a decrease in Replikin concentration and several years later a rebound increase associated in each case with a strain-specific rebound epidemic. The x-axis of FIG. 11 shows the year and the y-axis shows the Replikin concentration in Replikin sequences per 100 amino acids. Note that within FIG. 11 there is a separate graph for each strain responsible for each pandemic.
FIG. 12 is a graph illustrating an increase in Replikin concentration in spike and nucleocapsid coronavirus proteins preceding the SARS coronavirus epidemic of 2003. The x-axis indicates the year and the y-axis indicates the Replikin concentration. The appearance of the SARS outbreak and the eight countries involved in the outbreak is shown by the conical shaded area. The solid black symbols represent the mean Replikin concentration for spike coronavirus proteins and the vertical black bars represent the standard deviation of the mean. FIG. 12 illustrates a remarkable constancy of low coronavirus Replikin concentration between 1995 and 2001 in the spike proteins, followed by a dramatic increase in 2002, one year before the SARS epidemic appeared in 2003.
FIG. 13 is a graph comparing (1) the concentration of Replikin sequences observed in isolates of West Nile Virus having publicly available accession numbers on a year by year basis between 1999 and 2006 (with standard deviation bars for each Replikin concentration data point except 2000), (2) total morbidity reported in the United States on a year by year basis by the Center for Disease Control (total U.S. morbidity is the value denoted on the y-axis times 1000), and (3) total mortality resulting from WNV infection reported in the United States on a year by year basis by the Center for Disease Control (total U.S. mortality is the value denoted on the y-axis times 100). A correlation between Replikin concentration, morbidity and mortality is demonstrated.
FIG. 14 is a graph illustrating the mean concentration of Replikins observed in the VP1 protein of isolates of Foot and Mouth Disease Virus having publicly available accession numbers on a year by year basis between 1998 and 2006. Observed outbreaks of Foot and Mouth Disease in the United Kingdom (UK) are noted in 2001 and 2007.
FIG. 15 illustrates an increase in Replikin concentration in a Replikin Peak Gene in the pB1 genomic area of Equine Influenza one to two years in advance of epidemics of Equine Influenza. The graph illustrates that no increase in Replikin concentration in pA or pB2 genes of the equine influenza virus correlates with the increase in the Replikin concentration of the Replikin Peak Gene or with outbreaks of the virus.
FIG. 16 illustrates a magnification of the effect of increases in Replikin concentration on human mortality from H5N1 infections when Replikin concentration is observed in the Replikin Peak Gene (pB1 gene area) as compared to the polymerase gene or compared to the entire genome of the H5N1 virus. FIG. 16 is a graph of the data disclosed in Table 10. The graph demonstrates that an increase in Replikin concentration is magnified in its correlation with human mortality when restricted from changes in Replikin concentration in the entire genome to changes in Replikin concentration in the polymerase gene and magnified even more when restricted solely to the Replikin Peak Gene identified using the methods described herein. In FIG. 16, a correlation was established between human mortality and (1) mean concentration of Replikin sequences in the whole genome, (2) mean concentration of Replikin sequences in the polymerase gene, and (3) mean concentration of Replikin sequences in the Replikin Peak Gene (pB1 gene area) of H5N1 influenza strains. As Replikin concentration increased by these three measures, human mortality was observed to increase. However, while all three measures provided a correlation with human mortality, changes in the Replikin concentration in the polymerase gene correlated more significantly with human mortality, and changes in the Replikin concentration in the Replikin Peak Gene (pB1 gene area) of the H5N1 genome correlated still more significantly with human mortality. FIG. 16 suggests, therefore, that identification of Replikin Peak Genes within viral genomes improves identification and prediction of virulence and mechanisms of virulence using Replikin concentration data.
FIG. 17 illustrates a significant eight-fold increase in Replikin concentration in the pB1 gene area (Replikin Peak Gene) of isolates of H5N1 from 2003 through the first quarter of 2007 (that correlates with an increase in host mortality in humans), while no significant increase is observed in neighboring gene areas of the pB1 gene area, namely, the pA gene area and the pB2 gene area. FIG. 17 graphically compares percent human mortality from H5N1 infections in years 2005 through the first quarter of 2007 to mean concentration of Replikin sequences in (1) the pB1 gene area, (2) the pB2 gene area, and (3) the pA gene area, respectively, of H5N1 influenza strains isolated in 2003 through the first quarter of 2007. FIG. 17 illustrates a significant correlation between human mortality and the Replikin Peak Gene (pB1 gene area) of isolates of H5N1 influenza virus. No correlation is observed in neighboring gene areas of the pB1 gene area. In addition to the correlative aspect of the increase in Replikin concentration being related to percent mortality, FIG. 17 (graphically representing part of the data in Table 10) provides strong confirmation of the power and validity of the methodology of predicting changes in virulence and outbreaks of virus by monitoring changes in Replikin concentration.
 DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein, “animal” includes mammals, such as humans.
As used herein, the term “peptide” or “protein” refers to a compound of two or more amino acids in which the carboxyl group of one amino acid is attached to an amino group of another amino acid via a peptide bond. As used herein, “isolated” or “synthesized” peptide or biologically active portion thereof refers to a peptide that is, after purification, substantially free of cellular material or other contaminating proteins or peptides from the cell or tissue source from which the peptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized by any method, or substantially free from contaminating peptides when synthesized by recombinant gene techniques. An “encoded” protein, protein sequence, protein fragment sequence, or peptide sequence is a sequence encoded by a nucleic acid sequence that encodes the amino acids of the protein or peptide sequence with any codon known to one of ordinary skill in the art now or hereafter. It should be noted that it is well-known in the art that, due to redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon and still result in an identical amino acid sequence. As will be understood by one of skill in the art, a method of identifying a Replikin amino acid sequence also encompasses a method of identifying a nucleic acid sequence that encodes a Replikin amino acid sequence wherein the Replikin amino acid sequence is encoded by the identified nucleic acid sequence.
As used herein, a Replikin sequence is an amino acid sequence having about 7 to about 50 amino acids comprising:
    • (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;
    • (2) at least one histidine residue; and
    • (3) at least 6% lysine residues.
A Replikin sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine. A Replikin peptide or Replikin protein is a peptide or protein consisting of a Replikin sequence. A Replikin sequence may also be described as a Replikin sequence of about 7 to about 50 amino acids comprising or consisting of a Replikin motif wherein the Replikin motif comprises:
    • (1) at least one lysine residue located at a first terminus of said isolated influenza virus peptide and at least one lysine residue or at least one histidine residue located at a second terminus of said isolated influenza virus peptide;
    • (2) a first lysine residue located six to ten residues from a second lysine residue;
    • (3) at least one histidine residue; and
    • (4) at least 6% lysine residues.
      For the purpose of determining Replikin concentration, a Replikin sequence must have a lysine residue at one terminus and a lysine or a histidine residue at the other terminus.
The term “Replikin sequence” can also refer to a nucleic acid sequence encoding an amino acid sequence having about 7 to about 50 amino acids comprising:
    • (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;
    • (2) at least one histidine residue; and
    • (3) at least 6% lysine residues,
      wherein the amino acid sequence may comprise a terminal lysine and may further comprise a terminal lysine or a terminal histidine.
As used herein, a Replikin Peak Gene (RPG) or a Replikin Peak Gene Area (RPGA) are used interchangeably to mean a segment of a genome, protein, segment of protein, or protein fragment in which an expressed gene or gene segment has a highest concentration of continuous, non-interrupted and overlapping Replikin sequences (number of Replikin sequences per 100 amino acids) when compared to other segments or named genes of the genome. Generally, the gene or gene segment associated with a whole protein or protein-expressing gene is known as the Replikin Peak Gene and the gene or gene segment associated with a protein fragment is known as a Replikin Peak Gene Area. More than one RPG or RPGA may be identified within a gene, gene segment, protein, or protein fragment. An RPG or RPGA may have a terminal lysine or a terminal histidine, two terminal lysines, or a terminal lysine and a terminal histidine. An RPG or RPGA may likewise have neither a terminal lysine or a terminal histidine as long as it contains a Replikin sequence or Replikin sequences defined by the definition of a Replikin sequence, namely, an amino acid sequence having about 7 to about 50 amino acids comprising:
    • (1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;
    • (2) at least one histidine residue; and
    • (3) at least 6% lysine residues.
As used herein, “reservoir” is any source of Replikin sequences that may be shared with an influenza virus including any host of influenza virus, any food source of a host of the influenza virus, any vector of influenza virus, or any substance wherein the genetic information of the influenza virus may be shared, mingled, mixed, exchanged, or come into the proximity of the Replikin sequences of the reservoir.
As used herein, “different time periods” or “different time points” is any two time periods or points that may be differentiated one from another. For example, an isolate of virus isolated during the year 2004 is isolated in a different time period than an isolate of the same virus isolated during the year 2005. Likewise, an isolate of virus isolated in May 2004 is isolated in a different time period than an isolate of the same virus isolated in June 2004. When comparing Replikin concentrations of different isolates, it is preferred to use comparable time periods for comparison. For example, an isolate from 2004 is preferably compared to at least one other isolate from some other year such as 2002 or 2005. Likewise, an isolate from May 2004 is preferably compared to at least one isolate from some other month of some year, for example, an isolate from December 2003 or from June 2004. An isolate is any virus isolated from a natural source wherein a natural source includes, but is not limited to, a reservoir of a virus, a vector of a virus or a host of a virus. “Obtaining” an isolate is any action by which an amino acid or nucleic acid sequence within an isolate is obtained including, but not limited to, isolating an isolate and sequencing any portion of the genome or protein sequences of the isolate, obtaining any nucleic acid sequence or amino acid sequence of an isolate from any medium, including from a database such as PubMed, wherein the nucleic acid sequence or amino acid sequence may be analyzed for Replikin concentration, or any other means of obtaining the Replikin concentration of a virus isolated from a natural source at a time point.
As used herein, an “earlier-arising” virus or organism is a specimen of a virus or organism collected from a natural source of the virus or organism on a date prior to the date on which another specimen of the virus or organism was collected from a natural source. For viruses, a natural source includes, but is not limited to, a reservoir of a virus, a vector of a virus, or a host of the virus. A “later-arising” virus or organism is a specimen of a virus or organism collected from a natural source of the virus (including, but not limited to, a reservoir, a vector, or a host) or a natural source of the organism on a date subsequent to the date on which another specimen of the virus or organism was collected from a natural source.
As used herein, “emerging strain” refers to a strain of a virus identified as having an increased or increasing concentration of Replikin sequences in one or more of its protein sequences relative to the concentration of Replikins in other strains of such organism. The increased or increasing concentration of Replikins occurs over a period of preferably at least about six months, at least about one year or at least about three years, but may be a much shorter period of time for highly mutable viruses. An emerging strain of virus indicates an increase in virulence or replication.
As used herein, “bird” is any avian species including migratory and domestic birds, wherein said migratory and domestic birds includes, for example, chickens, ducks of all kinds, geese, pigeons, gulls, seabirds etc.
As used herein, “outbreak” is an increase in virulence, morbidity or mortality in a viral disease as compared to a baseline of an earlier occurring epidemiological pattern of infection in the same viral disease. One of ordinary skill in the art will know how to determine an epidemiological baseline. As used herein, “morbidity,” is the number of cases of a disease caused by the virus, either in excess of zero cases in the past or in excess of a baseline of endemic cases in the past. Therefore the baseline of endemic cases, in epidemiological terms, may, for example, relate to whether no or some cases were present in a geographic region in the immediate past. The past, in epidemiological terms, may mean more than one year and can mean several years or more as understood by one of ordinary skill in the art. The past may also mean less than one year as determined by one of ordinary skill in the art. In the case of annually-recurrent common influenza, for example, the baseline reflects an annual recurrence of common influenza.
As used herein, “mutation” refers to a change in the structure and properties of a virus or organism caused by substitution of amino acids. In contrast, the term “conservation” as used herein, refers to conservation of particular amino acids due to lack of substitution. A “point mutation” may refer to a change in a single amino acid residue or may refer to a change in a small number of amino acid residues.
As used herein, “replikin count” or “replikin concentration” refers to the number of Replikins per 100 amino acids in a protein, protein fragment, virus, or organism. A higher Replikin concentration in a first strain of a virus or organism has been found to correlate with more rapid replication of the first virus or organism as compared to a second, earlier-arising or later-arising strain of the virus or organism having a lower Replikin concentration.
As used herein a “Replikin Scaffold” refers to a series of conserved Replikin peptides wherein each of said Replikin peptide sequences comprises about 16 to about 34 amino acids, and preferably about 27 to about 33 amino acids and further comprises: (1) a terminal lysine and optionally a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and optionally a histidine immediately adjacent to the terminal histidine; (3) a lysine within 6 to 10 amino acid residues from another lysine; and (4) about 6% lysine. “Replikin Scaffold” also refers to an individual member or a plurality of members of a series of Replikin Scaffolds.
In an influenza virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a preferred embodiment about 28 to about 30 amino acid residues. In a White Spot Syndrome Virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a more preferred embodiment about 29 to about 31 amino acid residues. In a Taura Syndrome Virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a more preferred embodiment about 29 to about 33 amino acid residues.
Predicting Virulence by Determining Replikin Concentration in Viral Reservoirs, Vectors and Hosts
The inventors provide herein methods of predicting outbreaks of influenza by monitoring the presence and/or concentration of Replikin sequences both throughout the virus and in specific highest concentrations of Replikins in areas designated as Replikin Peak Genes, Replikin Peak Gene Areas and/or Replikin Scaffold sequences in WSSV and TSV in shrimp and influenza viruses.
Before identification of the Replikin structure in virus proteins there existed no quantitative measure of virus structure that correlated quantitatively with rapid replication or that gave advance warning of virus outbreaks or emerging virus disorders.
Since the identification of the Replikin structure, correlation between increased concentrations of Replikin sequences and increased replication and virulence has been observed in a range of viruses and organisms. These observations are made more accurate by the present isolation in silico of Replikin Peak Genes. A Replikin Peak Gene includes the area in a genome, protein, or protein fragment that has the highest concentration of Replikin sequences. While increased concentration of Replikin sequences in the genome of a virus offers both advance warning and new targets for developing effective methods of predicting and treating viral outbreaks, identification of an increase in concentration of Replikin sequences in a Replikin Peak Gene of a genome or protein heightens the predictive capacity of the change in Replikin concentration and the efficacy of new targets.
For example, more precise predictions of increased virulence are now available through identification of a Replikin Peak Gene in, among other viruses, the H5N1 strain of influenza (FIGS. 3 and 8-10), the H3N8 strain of influenza that causes equine influenza (FIG. 15), West Nile Virus (FIG. 13), Foot and Mouth Disease Virus (FIG. 14) and White Spot Syndrome Virus (Example 1). In these and other viruses, increased concentration of Replikin sequences in the whole genome, in a protein of the genome, or in a Replikin Peak Gene of the genome offer both advance warning and new targets for developing effective methods of predicting and treating viral outbreaks.
By monitoring changes in concentrations of Replikin sequences in viral genomes generally, emerging viral diseases can be identified in virus reservoirs and vectors in advance of their appearance in animal or human hosts. Identification of the emerging viruses and the Replikin sequences within the virus genome allows for appropriate, advance control efforts, including isolation and quarantine, and provides sufficient time for the synthesis and testing of vaccines specific to the sequences of the emerging virus.
Replikin sequences that had previously been shown to pass between several strains of the same virus, as in the H1N1, H2N2, H2N3 and H5N1 strains of influenza A virus, have now been found to pass between different viruses as well as different hosts, as though the Replikin structure is the key infectious and lethal unit of the disease and the virus is the carrier or vector of this infectious and lethal unit. Viewing the Replikin structure as an infectious unit of disease then allows the host organism or the virus itself to be viewed (at any given point in the infectious cycle) as a host, a reservoir, or a vector for Replikin units shared by viruses, host organisms, and vector organisms in the infectious pathway of the disease.
Identification of Replikin sequences as infectious units (and providing particular areas in the genome, i.e. Replikin Peak Genes, where those infectious units are correlated most significantly with virulence) has allowed the inventors to focus attention on the geographic area (see FIG. 10), the host (see FIG. 9), and the particular area of the genome of a virus (see FIG. 8) wherein virulence mechanisms are located and thereby increased virulence may be identified and predicted. Because the inventors have provided a method of focusing on particular units of a viral genome, the skilled artisan will understand the importance of looking for Replikin sequences in any portion of the life cycle or infectious pathway of a virus. For example, as described herein, the skilled artisan will understand that predictive and virulence-related Replikin sequences (or concentrations of Replikin sequences in Replikin Peak Genes) may be identified in reservoirs of influenza virus such as in WSSV and TSV. The skilled artisan will further understand that predictive and virulence-related Replikin sequences or Replikin Peak Genes may be identified in vectors of the influenza virus. The skilled artisan will additionally understand that predictive and virulence-related Replikin sequences or Replikin Peak Genes may be identified in hosts of the influenza virus or in any other place where viral genes may be located or wherein viral genetic information may encounter genetic information of other strains of virus, of other virus species, of vectors of the virus, of hosts of the virus, or of food sources of hosts of the virus. This method of locating relevant Replikin sequences finds support in the inventors' disclosure of data providing insight into what sequence changes in influenza since 1917 were due to “drift” or “shift” or, on the other hand, what sequence changes in influenza since 1917 were due to conservation, storage in “reservoirs,” and reappearance in both intermediate vectors and eventual animal and human hosts. The data has shown that the epidemic-related increase in Replikin concentration is not due to the duplication of existing Replikins in the hemagglutinin of the emerging strain, but instead is due to the reappearance of at least one Replikin composition from 1 to up to 59 years after its disappearance, plus (in the A strains only) the emergence of new strain-specific Replikin compositions. See U.S. Pat. No. 7,189,800 issued Mar. 13, 2007 (Tables 3-6).
In another embodiment of the invention, an automated prediction of an outbreak of influenza virus is made by (1) measuring the Replikin concentration in a WSSV or TSV isolate structure, or other reservoir virus structure (2) comparing the measured concentration to the Replikin concentration determined at a previous time point in the same virus structure, and (3) observing an increase in Replikin concentration in that virus structure. If an increase has occurred, an outbreak within about one to about three years following the latest time point is predicted. In a further embodiment, the increase from one time point to another time point is statistically significant.
The magnitude of a Replikin increase in a virus may be a quantitative component of predicting an outbreak. For example, in H5N1 influenza virus, a two-fold (200%) increase in Replikin concentration predicted an outbreak of the virus in humans. See FIG. 16 and Example 9. Further, an eight-fold (800%) increase in the Replikin Peak Gene of the virus predicted the same outbreak, which included new morbidity and increased mortality in infections from the virus. See FIG. 17 and Example 9.
Replikin Concentration in WSSV and TSV Predicts Human Influenza Outbreaks
The present inventors have discovered that two viruses, White Spot Syndrome Virus (WSSV) (also known as white spot baculoform virus) and Taura Syndrome Virus (TSV), both global lethal pathogens for shrimp, are reservoirs for the peptide building blocks of influenza virus, including the H5N1 strain of influenza virus. The concentration of Replikins in WSSV, TSV, and in influenza has been shown to be related to rapid replication and epidemics in each of these viruses. An increase in the concentration of Replikin sequences has been correlated with the last three influenza pandemics of 1918, 1957 and 1968. See FIG. 11. Quantitative determination of the concentration of virus Replikins has made it possible to predict in advance recent influenza outbreaks including outbreaks of the H5N1 strain of influenza. Quantitative determination of the concentration of Replikins in viruses for which nucleic acid or protein sequences are available may now be automated using the proprietary FluForecast®, which is available from Replikins, LLC, Boston Mass.
The H5N1 virus recently has been responsible for huge poultry losses in many countries and for several hundred human cases with approximately 50% mortality. While migratory waterfowl are known to transport H5N1 influenza virus globally, no reservoirs for the virus had yet been identified. The inventors, however, have now identified shrimp as a reservoir for homologous Replikin sequences identified in White Spot Syndrome Virus (WSSV) and Taura Syndrome Virus (TSV). The homologous Replikin sequences have been identified in both shrimp and influenza virus and in particular in the H5N1 “bird flu” strain of influenza virus in both birds and humans.
Beginning with evidence of a sharing of homologous Replikin sequences among the three viruses, namely, WSSV, TSV, and strains of influenza virus including H5N1, the inventors identified a correlation between an increase in Replikin concentration in WSSV and/or TSV and an increase in virulence (and a concomitant increase in Replikin concentration) in the H5N1 strain of influenza virus. The inventors further identified homologous Replikin sequences shared within and between the virus strains including Replikin Scaffold sequences, the presence of which have been associated with high virulence, epidemics and pandemics.
Using these correlations and observations, the inventors have now devised a method of predicting an outbreak of influenza virus comprising:
  • (1) obtaining a plurality of isolates of White Spot Syndrome Virus or a plurality of isolates of Taura Syndrome Virus wherein at least one of said isolates of White Spot Syndrome Virus is isolated about six months to about 3 years later than at least one other of said isolates of White Spot Syndrome Virus, or wherein at least one of said isolates of Taura Syndrome Virus is isolated about six months to about 3 years later than at least one other of said isolates of Taura Syndrome Virus;
  • (2) analyzing the amino acid sequence of at least one encoded protein or protein fragment or a combination of encoded proteins and/or protein fragments in each isolate of the plurality of isolates of White Spot Syndrome Virus or the plurality of isolates of Taura Syndrome Virus for the presence and concentration of encoded Replikin sequences;
  • (3) comparing the concentrations of encoded Replikin sequences in the at least one protein or protein fragment or in the combination of proteins or protein fragments in each isolate of the plurality of isolates of White Spot Syndrome Virus or in each isolate of the plurality of isolates of Taura Syndrome Virus;
  • (4) identifying an increase in the concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus or in said plurality of isolates of Taura Syndrome Virus over at least one time period of about six months or greater, at least about one year or greater, or at least about three year to five years; and
  • (5) predicting an outbreak of influenza within about one to about three years following said identified increase in the concentration of Replikin sequences in said isolates of White Spot Syndrome Virus or in the concentration of Replikin sequences in said isolates of Taura Syndrome Virus.
Prediction of an outbreak of influenza may further comprise:
  • (1) identifying at least one Replikin Scaffold sequence present in at least one isolate of the plurality of isolates of White Spot Syndrome Virus identified as having an increase in the concentration of Replikin sequence as compared to at least one other isolate of the plurality of isolates of White Spot Syndrome Virus over a time period of about six months or greater, or identifying at least one Replikin Scaffold sequence present in at least one isolate in the plurality of isolates of Taura Syndrome Virus identified as having an increase in the concentration of Replikin sequences as compared to at least one other isolate of the plurality of isolates of Taura Syndrome Virus over a time period of about six months or greater;
  • (2) identifying at least one Replikin Scaffold sequence present in at least one isolate of a strain of influenza virus; and
  • (3) predicting the strain of influenza virus in which the at least one Replikin Scaffold sequence is identified as the strain of virus that will likely cause the predicted outbreak of influenza virus wherein a Replikin Scaffold is a peptide having about 16 to about 34 amino acids comprising (a) a terminal lysine and optionally a lysine immediately adjacent to the terminal lysine; (b) a terminal histidine and optionally a histidine immediately adjacent to the terminal histidine, (c) a lysine within about 6 to about 10 amino acids of another lysine; and (d) at least 6% lysines.
In an influenza virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues and in a preferred embodiment about 28 to about 30 amino acid residues. In a White Spot Syndrome Virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a more preferred embodiment about 29 to about 31 amino acid residues. In a Taura Syndrome Virus, a Replikin Scaffold may refer to a Replikin peptide sequence comprising about 16 to about 34 amino acid residues, and in a more preferred embodiment about 29 to about 33 amino acid residues.
The shrimp industry understands that a first clue that virus infection from WSSV or TSV has occurred in shrimp ponds is observation of the death of migratory birds on the periphery of the pond. This observation suggested to the inventors that shrimp and birds shared some pathogenic mechanism related to shrimp infection with WSSV and/or TSV. By analyzing Replikin concentrations in publicly available amino acid sequences of WSSV, TSV and H5N1 isolates of virus using FluForecast® software available through Replikins, LLC, Boston, Mass., USA, the following findings were obtained, which demonstrated that the shrimp viruses WSSV and TSV serve as a reservoir of Replikin peptide building blocks for H5N1 and other influenza strains:
    • 1) The shrimp viruses WSSV and TSV were found to contain Replikin peptide sequences and an increase in concentration in these sequences was correlated with specific outbreaks of the virus.
    • 2) These shrimp virus sequences were found to be related in structure to Replikin peptide sequences in H5N1 and other strains of influenza virus that had been specifically associated with virulence, epidemics, pandemics and human mortality in influenza.
    • 3) Shrimp WSSV Replikins increased markedly in concentration in the year 2000, just before an increase in Replikin concentration in H5N1 that directly preceded H5N1 outbreaks in chickens, other birds, and humans between 2001-2006. The increase in shrimp virus Replikin concentration was not trivial. In shrimp WSSV, which in dormant states was found to have well less than 10 Replikin sequences within 100 amino acids, Replikin concentration reached the remarkable height of 103.8 in an isolate in 2000 and a mean of 97.6 among all isolates with publicly available protein sequences. See Example 4. The remarkable Replikin concentration of 103.8 is comparable only to the highest Replikin concentration so far observed in any organism in nature, namely, P. falciparum, which has the highest Replikin concentration to date—111 Replikin sequences observed per 100 amino acid residues. P. falciparum is a cause of malaria and has been observed to replicate 11,000 times in 48 hours when passing in its life cycle from the liver to blood in its host.
    • 4) Of the new shrimp Replikins that appeared in 2000, the percent that were short peptides was increased compared to dormant years. Short Replikins previously have been found to be related to high virulence and high mortality in the host, whether animal or man. See. e.g., U.S. application Ser. No. 10/860,050, filed Jun. 4, 2004.
    • 5) These short shrimp virus Replikins share structures with short Replikins in both H5N1 and other influenza strains going back 89 years to the great pandemic of 1918.
    • 6) Replikin Scaffold sequences were identified in proteins of WSSV and TSV that were highly homologous with Replikin Scaffold sequences in highly virulent strains of influenza virus including the H5N1 strain.
The occurrence of the major WSSV outbreak in the year 2000 places WSSV temporally in line with the influenza outbreaks that occurred successively in other hosts: (1) in ducks in 2004-2005; (2) in chickens in 2005-2006; and (3) in humans in 2006-2007. See FIG. 9. Each outbreak was preceded by major increases in the respective Replikin concentrations of the virus and in the Replikin concentrations of their Replikin Peak Genes. See, e.g., U.S. Appln. Ser. No. 60/898,097, filed Jan. 30, 2007, FIGS. 1-4. An outbreak of TSV in 2000 likewise was temporally in line with the influenza outbreaks of 2001-2007.
The mortality rate of shrimp for the WSSV shrimp virus is 80-100% and, as shown above, the Replikin concentration during outbreaks has exceeded 100 Replikin sequences per 100 amino acid residues. The Replikin concentrations and the mortality rates in H5N1 have not been as high in ducks and chickens. Replikin concentrations of twenty in the Replikin Peak Gene of H5N1 have been observed in humans in Indonesia along with mortality rates of approximately 80% in a small number of humans in 2006-2007. See U.S. application. Ser. No. 11/755,597, filed May 30, 2007, pages 36-39.
While Applicants do not wish to be constrained by theory, this evidence may reflect migration or vectoring of Replikin sequences between species, or may reflect successive independent stimulation of influenza strains in different hosts, or both, or even some other yet-to-be-determined mechanism. Data that supports migration of Replikin sequences between different strains of influenza virus rather than independent synthesis has been reported in previous patent applications by the Applicants (see, e.g., U.S. Pat. No. 7,189,800 and U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006), but the present evidence of relation of shrimp viruses to influenza viruses is the first evidence of inter-virus species migration of Replikins. In effect, the “infectious unit” of a virus may not be the whole virus but, instead, a relatively small constituent of the virus structure, namely Replikin sequences.
Replikin Scaffold Sequences in WSSV, TSV, and Influenza
Replikin Scaffold sequences so far identified in WSSV begin with KK and end with H. The length of the Scaffold Sequence is about 27 to 31, which is similar to the about 28 to about 29 amino acid Replikin Scaffolds generally observed in the H5N1, H1N1, H2N2, H2N3, and H7N7 strains of influenza virus. The following Replikin Scaffold repeats were identified in Accession No. AAL89390, which discloses a 2000 isolate of WSSV.
(SEQ ID NO: 1)
K66KNVKSAKQLPHLKVHLDVKSAKQLPHLKVH96
(SEQ ID NO: 2)
K160KNVKSAKQLPHLKVHLDVKGVKQLLH186
(SEQ ID NO: 3)
K239KNVKSAKQLPHLKVLLDVRGAKQLPH265
(SEQ ID NO: 3)
K303KNVKSAKQLPHLKVLLDVRGAKQLPH329
(SEQ ID NO: 4)
K397KNVKSAKQLPHLKVLLDVRGAKQLPHLKVH427.
In the above-listed Replikin Scaffolds, the orderly substitution of H for L in positions 81 and 175 and K for R in positions 85 and 179 both result in an increase in the number of Replikins per 100 amino acids, i.e. in an increase in the Replikin concentration. The phenomenon of Replikin scaffolding also has been observed in P. falciparum (malaria). The substitution of lysines and histidines resulting in increased Replikin concentration and increased virulence also has been observed by the applicants in H5N1 in a Replikin Scaffold in China (See U.S. application Ser. No. 11/755,597, filed May 30, 2007, Table 1). The homologous structural properties in shrimp WSSV and TSV and influenza provide continuing support for the structural relationship between Replikin sequences in WSSV and TSV and influenza viruses.
Accordingly one aspect of the invention provides a method of predicting an influenza outbreak by correlating an increase in Replikin concentration in shrimp WSSV and/or TSV over Replikin concentration in previous isolates of WSSV and/or TSV with an increase in virulence and/or replication of influenza viruses.
Presence of Replikin Scaffold is Predictive of Epidemics
The inventors have established that the presence of Replikin Scaffolds in influenza strains is predictive of epidemics. As such, in addition to the total number of Replikins in a virus, the structure of each Replikin through time is informative. Table 1 shows a Replikin Scaffold first observed in a goose infected with influenza in 1917 (Goose Replikin). Constant length, constant lysines at the amino terminal and histidine residues at the carboxy terminal were conserved in different strains in a fixed scaffold for decades. Homologues of the Goose Replikin appeared from 1917 through 2006 in strains including each strain responsible for the three pandemics of 1918, 1957, and 19681, H1N1, H2N2 and H3N2, and with further substitutions between H1N2, H7N7, H5N2 and H5N1.
TABLE 1
Replikin Scaffold showing ordered substitution in the 89 year conservation of influenza
virus Replikin peptides related to rapid replication, from a 1917 goose influenza Replikin
and the 1918 human pandemic Replikin to 2006 H5N1 “Bird Flu” homologues.
(SEQ ID NOS: 368-430, respectively, in order of appearance)
|<--------------29 Amino Acids------------→| Year Strain
kkgtsypklsksytnnkgkevlvlwgvhh 1917 H1N_Influenza Goose Replikin
Figure US08050871-20111101-C00001
 		1918 1930 1933 H1N1 Human Influenza Pandemic H1N1 H0N1
kkgtsypklsksytnnkgkevlvlwgvhh 1976 H1N1
kkgtsypklsksytnnkgkevlvlwgvhh 1977 H1N1
Figure US08050871-20111101-C00002
 		1979 1980 H1N1 H1N1
kkgtsypklsksytnnkgkevlvlwgvhh 1980 H1N1
Figure US08050871-20111101-C00003
 		1981 H1N1
kkgtsypklsksytnnkgkevlvlwgvhh 1981 H1N1
kkgtsypklsksytnnkgkevlvlwgvhh 1985 H1N1
Figure US08050871-20111101-C00004
 		1991 1992 1996 1996 1997 1998 1999 2000 2001 2002 1999 2000 2001 H1N1 H1N1 H1N1 H1N1 H1N1 H1N1 H1N1 H1N1 H1N1 H1N1 H1N2 Influenza H1N2 H1N2
Figure US08050871-20111101-C00005
 		2001 2002 2002 1957 1957 1968 1979-2003 2002 1959 1975 1981 1983 1988 1996 1997 1998 1999 2000 2001 2001 2002 2002 H1N2 H1N2 H1N2 H2N2 Human Influenza Pandemic H2N2 Human Influenza Pandemic H3N2 Human Influenza Pandemic H7N7 Influenza H5N2 Influenza H5N1 Influenza (Scotland) H5N1 (Wisconsin) H5N1 (Minnesota) H5N1 (Pennsylvania) H5N1 (Scotland) H5N1 (China) H5N1 (China) H5N1 (China) H5N1 (China) H5N1 (China) H5N1 (China) H5N1 (China) H5N1 (China) H5N1 (Thailand)
Figure US08050871-20111101-C00006
 		2002 2003 2003 2003 2003 2004 2004 2004 2004 2004 2004 2004 2004 2005 2006 2006 H5N1 (Vietnam) H5N1 (Vietnam) H5N1 (Thailand) H5N1 (Sindong, China) H5N1 (China) H5N1 (Vietnam, highly pathogenic) H5N1 (Vietnam, highly pathogenic, gull) H5N1 (Vietnam highly pathogenic) H5N1 (Thailand, highly pathogenic) H5N1 (Thailand, highly pathogenic) H5N1 (China, highly pathogenic) H5N1 (China, highly pathogenic, goose) H5N1 (Japan) HSN1 (Turkey) HSN1 (China, Anhui) HSN1 (Indonesia, highly pathogenic)
* Residues identical to Goose Replikin amino acids are unshaded; amino acid substitutions are shaded lightly and darkly to show scaffold pattern across years and strains. Substitution at position 24 in 2004 and 2006 H5N1, 1957 H2N2, 1968 H3N2 and H7N7 are boxed.
Table 1 illustrates the history, by year or smaller time period, of the existence in the protein structure of the Goose Replikin and its homologues in other influenza Replikins. Table 1 further illustrates the history of amino acid substitutions in those homologues and the conservation of certain amino acids of the Replikin structure that are essential to the definition of a Replikin and the function of rapid replication supplied by Replikins.
A review of Table 1 illustrates that if random substitution of amino acids were to occur in virulent strains of influenza from 1917 through the present, certain framework amino acids of the Goose Replikin would not be conserved from year to year in strains in which epidemics occurred. However, contrary to what would result from random substitution, virulent strains of influenza from year to year consistently contain conserved amino acids at those positions that define a Replikin. That is, if a substitution were to occur in one of the amino acids that define a Replikin, e.g. lysine or a histidine, the definition of the Replikin would be lost. Nevertheless, the Replikin sequence is conserved over more than 89 years. Thus, since there is conservation of certain amino acids over decades, substitution cannot be said to be completely at random. The fact that substitutions do occur in amino acids that are not essential to the definition of a Replikin (i.e., amino acids other than lysines or histidines) demonstrates the importance of the Replikin and the Replikin Scaffold in the pathogenicity of the strain.
It may be further noted from Table 1 that when substitutions do occur, they are seen to occur at certain apparently preferred positions of the Replikin Scaffold. Table 1 illustrates recurring substitutions at positions 1, 3-24 and 26-27. Further, while substitutions occur throughout these positions, a lysine continues to exist at a position 6 to 10 amino acids from a second lysine (which has not been substituted in these virulent strains).
Even when there is a substitution of a lysine position within the 29 amino acid stretch, as is seen in 1957, when K at position 11 shifts to position 10, that new position is maintained until 2005. Additionally, YP (at positions 6-7), SY (at positions 12-13), N (at position 15), and LVLWG (SEQ ID NO: 5) (at positions 22-26) conserve the homologous structure of the Replikin Scaffold with few exceptions.
In the 1997H5N1 Hong Kong epidemic, the human mortality rate was approximately 27%. In 2004, of the fifty-two people reported to have been infected by H5N1 in Asia, approximately 70% died. Nine of the eleven people infected in Vietnam from Dec. 28, 2004 to Jan. 27, 2005 died. Although the virulence of the virus appears to have increased, any changes thought to be required for further spread from human to human, had been thought not yet to have occurred. However, the inventors observed substitutions in three H5N1 Replikin amino acid residues at position numbers 18, 24 and 28 of the Goose Replikin scaffold from isolates in Vietnam, Thailand and China in 2004 (see Table 1). Substitution at position number 24 has not occurred since the appearance of H5N1 in 1959 but was present in the last two influenza pandemics caused by other strains, H2N2 in 1957 and H3N2 in 1968, together responsible for over two million human deaths, and in a recent virulent epidemic caused by H7N7 (see Table 1). These data on substitution, combined with the rising Replikin concentration shown in FIG. 4, and the past correlation of such Replikin data with pandemics, predicted the small outbreak of H5N1 in humans in 2006 and 2007.
It is important to note that an extra K has also appeared in the Replikin Scaffold of a 2006 strain of H5N1 in China (Anhui). This presence of an extra K both produces and signals an increase in the Replikin count within the Replikin Scaffold. The 2006 China (Anhui) strain has a Replikin count of 6.6 (as discussed below). A Replikin count of 6.6 is the highest ever observed for an H5N1 strain and is comparable in the entire A strain of influenza only to the Replikin count of the influenza strain that caused the 1918 Pandemic.
Homologous Replikin Scaffold Sequences in Influenza, WSSV, and TSV
The inventors have further established a relationship between virulent influenza virus and WSSV and TSV in the Replikin Scaffold portions of the viruses as may be seen in Table 2 below. Although there is extensive substitution, several short Replikins of the Shrimp White Spot Syndrome Virus demonstrates significant homologies to the influenza virus Replikin sequences, especially with regard to length and key lysine (k) and histidine (h) residues. Similar, but less extensive, homologies are seen in Taura Syndrome Virus. These homologies suggest that the sequences are derived from a shared reservoir and/or that similar mechanisms of Replikin production are used in both virus groups.
TABLE 2
Shrimp White Spot and Taura Syndrome Scaffolding
Figure US08050871-20111101-C00007
 		(SEQ NO: 379) (SEQ NO: 380) (SEQ NO: 6) (SEQ NO: 7) (SEQ NO: 398) (SEQ NO: 399) (SEQ NO: 396) (SEQ NO: 397) (SEQ NO: 400) (SEQ NO: 401) (SEQ NO: 8) (SEQ NO: 9) 1971 2002 2000 2000 1968 1979-2003 1957 1957 2002 1959 2000 2005 H1N_ Influenza goose peptide H1N1 Swine Influenza Shrimp White Spot Syndrome Virus Shrimp White Spot Syndrome Virus H3N2 Human Influenza Pandemic H7N7 Influenza H2N2 Human Influenza Pandemic H2N2 Human Influenza Pandemic H5N2 Influenza H5N1 Influenza Taura Syndrome Virus Taura Syndrome Virus
Residues identical to original 1917 Goose Replikin residues are shown in
Figure US08050871-20111101-C00008
Amino acid substitutions in
Figure US08050871-20111101-C00009
 and
Figure US08050871-20111101-C00010
In addition, since many species, including but not limited to swine and birds, are known to provide animal “reservoirs” for human influenza infection, marine forms such as the shrimp virus can now be examined, with early warning diagnostic benefits for outbreaks such as swine flu and bird flu. While similarities of some influenza viruses were noted between species, and the transfer of these viruses interspecies was known, there was no previous quantitative method to gauge virus activity. The activity of Replikins in shrimp can now be monitored constantly for evidence of increased viral replication rate and thus emergence of epidemics that are likely to transfer to other species.
A related example of virus reservoir activity in which the Replikin concentration was increased prior to an outbreak was observed in corona viruses as a group. The Replikin concentration of the corona virus group increased markedly in 2002 before the outbreak of one of its members, SARS, in 2003. See FIG. 12.
With high mortality for its shrimp host, White Spot Syndrome Virus can now have its Replikins examined as earlier forms of virus Replikins, or as parallel morphological branches, that in either case may act as reservoirs for bird and animal Replikins such as those in influenza viruses. The diagnostic and preventive uses of these Replikin findings in shrimp follow as they do in influenza and for other organisms containing Replikins.
Advanced forecasts of influenza virus outbreaks are now conveniently achievable with Replikin concentration analysis using the proprietary software FluForecast® from Replikins, LLC, Boston, Mass. Such forecasts now may permit time for preventive public health measures to be mobilized and safer strain-specific vaccines to be synthesized, tested, and mass produced.
Replikin Repeats as a Mechanism for High Replikin Concentrations
The presence of repeat sequences of the Replikins of the nucleocapsid protein of shrimp White Spot Syndrome Virus (WSSV) accounts for the unusually high Replikin concentration of 103.8. This Replikin concentration is much higher than the Replikin concentration of for example, influenza viruses, which usually range from less than 1 up to 5 or 7 and comparable, as discussed above, with P. falciparum. Interestingly, while the shrimp White Spot Syndrome pathogen is a virus, and the P. falciparum is a trypanosome, both spend an essential part of their reproductive cycles in red blood cells, an unusual host cell whether in shrimp (White Spot Syndrome Virus) or man (malaria), both are fulminating, rapidly-replicating diseases with high mortality rates of their hosts, and both appear to use the same methods of increasing their high Replikin concentrations to such record highs, namely, Replikin repeats and Replikin overlap.
As illustrated in Table 3, examples of Replikin repeats and Replikin overlaps were found by the applicants in the above nucleocapsid protein of the shrimp White Spot Syndrome Virus. 497 Replikins were observed in the White Spot Syndrome Virus. Of those 497, the Replikins illustrated below in Table 3 were selected for their short sequences and high concentration of lysines which, as demonstrated throughout this application, is associated with high mortality. The chosen sequences are easier and less expensive to synthesize than the longer sequences that are not included in Table 3.
Table 3 illustrates intramolecular Replikin repeats and Replikin overlap in shrimp White Spot Syndrome Virus (WSSV) nucleocapsid protein (VP35) gene with a Replikin concentration (number of Replikins per 100 amino acids) of 103.8 (497 total Replikins per 479 amino acids). The nucleocapsid protein reportedly possesses thymidine kinase and thymidylate kinase activity.
TABLE 3
Intramolecular Replikin repeats and Replikin overlap in shrimp White Spot Syndrome Virus (WSSV) nucleocapsid protein (VP35) gene with Replikin concentration of 103.8 Individual Replikins at Different Positions in the same Molecule, in order of appearance in the sequence
Figure US08050871-20111101-C00011
 		(SEQ ID NO: 10) (SEQ ID NO: 11) (SEQ ID NO: 12) (SEQ ID NO: 10) (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 13) (SEQ ID NO: 13) (SEQ ID NO: 13) (SEQ ID NO: 13)
*Note in the shrimp virus the repeated use of identical whole Replikin sequences (underlined) and partial Replikin sequences (shaded) in different positions in the one molecule (each amino acid is numbered according to its order in the sequence).
Replikin concentration was determined for the ribonucleotide reductase gene in WSSV from Accession No. AAL89390. Accession No. AAL89390 discloses the amino acid sequence of ribonucleotide reductase translated from the total genome of a year 2000 isolate of White Spot Syndrome Virus made publicly available at Accession No. NC 003225.1. The Replikin concentration, as discussed above, was an unusually high at 103.8. Additionally, the Replikin concentration of the Replikin Peak Gene of the protein was even higher at 110.7. The Replikin concentration of the Replikin Peak Gene was determined by dividing the number of Replikin sequences identified in the segment of the protein containing the highest concentration of Replikin sequences, 497 Replikin sequences, by the total amino acid length of the Replikin Peak Gene, 449 amino acids, to arrive at 110.7 Replikin sequences per 100 amino acids. See Example 1.
The amino acid sequence of the protein publicly available at Accession No. AAL89390 is of particular interest because it demonstrates an overlapping of Replikin sequences that result in very high Replikin concentrations. The very large number of Replikin sequences present in the genome of the WSSV serves as a reservoir or vector for individual Replikins that are transferred or, via some other mechanism, otherwise appear in virulent strains of the influenza virus. This transfer or appearance of homologous Replikin sequences is seen in the Replikin Scaffold of the influenza virus and the Replikin Scaffold of the Shrimp White Spot Virus.
The amino acid sequence disclosed at Accession No. AAL89390 was further observed to contain significant Replikin Scaffold sequences. The presence of Replikin Scaffold sequences in Accession No. AAL89390 was not unexpected since Replikin Scaffolds are frequently present in viral genomes when the Replikin concentration is observed to move above 3 to 4 Replikin sequences per 100 amino acids. The presence of a Replikin Scaffold and a Replikin concentration above 3 or 4 correlates with viral outbreaks or epidemics and is another clear association with the rapid replication states of the isolated virus.
Replikin Concentration Correlates with 20th Century Influenza Pandemics
As discussed above, Replikin concentration has been correlated with virulence in each influenza pandemic of the 20th century. See FIG. 11. This shared correlation between Replikin concentration and outbreaks and mortality in WSSV and TSV and influenza provides evidence that the Replikin structure is transferable or repeatable between these viruses.
The correlation between Replikin concentration and influenza outbreaks is well established and unmistakable. Within the last century there have been three influenza pandemics, each strain specific: H1N1 in 1918; H2N2 in 1957; and H3N2 in 1968. In the reported amino acid sequences of the strains responsible for each pandemic, there is a strain-specific increase in the Replikin concentration correlated with the major pandemic within the strain, followed by a decrease in Replikin concentration and several years later a rebound increase associated in each case with a strain-specific rebound epidemic. The x-axis of FIG. 11 shows the year and the y-axis shows the Replikin concentration in Replikin sequences per 100 amino acids for each strain responsible for each pandemic. Note that within FIG. 11 there is a separate graph for each of the three strains.
FIG. 11 demonstrates that frequently a one to three year stepwise increase is observed before Replikin concentration reaches a peak. This stepwise increase precedes the occurrence of an epidemic, which occurs concurrently with the Replikin peak. Thus, the stepwise increase in concentration of a particular strain is a signal that a particular strain is the most likely candidate to cause an epidemic or pandemic.
H5N1 Influenza Replikin Concentration Correlates with Epidemics
Replikin concentration has also been correlated with virulence in each outbreak of H5N1 influenza (“Bird Flu”) virus between the initial outbreak of 1997 and the present. FIG. 4, for example, illustrates a correlation between each outbreak of H5N1 between 1997 and 2004. The x-axis in FIG. 4 indicates the year and the y-axis indicates the Replikin concentration (number of Replikins per 100 amino acids). The time periods for each epidemic of H5N1 are denoted in FIG. 4 by E1, E2 and E3. In H5N1 influenza, an increasing strain-specific Replikin concentration (+/−SD) from 1995 to 1997 preceded the Hong Kong H5N1 epidemic of 1997 (E1); an increase from 1999 to 2001 preceded the epidemic of 2001 (E2); and an increase from 2002 to 2004 preceded the epidemic in 2004 (E3). A decline in 1999 occurred with the massive culling of poultry in response to the E1 epidemic in Hong Kong.
H5N1 Influenza Conservation of Replikin Scaffold in Highly Virulent Isolates
There is concern that current high mortality H5N1 “bird flu” in several countries may represent an early phase of an overdue influenza pandemic. Analysis of Replikin concentration changes in H5N1 have suggested that H5N1 virulence is continuing to increase. A 2006 report nevertheless suggested that in the first probable person-to-person transmission of H5N1, “sequencing of the viral genes identified no change in the receptor-binding site of hemagglutinin or other key features of the virus. The sequences of all eight viral gene segments clustered closely with other H5N1 sequences from recent avian isolates in Thailand.” Phylogenetic analysis suggested that from the absence of evidence of “reassortment with human influenza viruses” that H5N1 is not a new variant. However, the inventors disclosed in 2006, three changes in a specific H5N1 protein sequence at sites which had not been changed in the last two H5N1 epidemics and in fact had been conserved since 1959. See U.S. Prov. Appln. Ser. No. 60/808,944, filed May 30, 2006.
The results set forth by the inventors in 2006 showed that 2005-2006 virus data indicated clearly that 1) the mortality rate of human H5N1 was increasing markedly, and that 2) the first country in which this would be clinically realized would be Indonesia. Bayu Krisnamurthi, the head of Indonesia's avian flu control commission, reported in June 2007 the clinical realization of both of these two predictions (Canadian Press, Jun. 6, 2007). In his comments to reporters, Dr. Krisnamurthi stated that recent changes in the H5N1 virus seem to be increasing its rate and ease of transmission from birds to humans. The World Health Organization (WHO), which has not yet implemented Replikin concentration analysis and tracking that had predicted the increase in rate and transmission in 2006, reported that they had no evidence of these changes.
Recent discovery of the Replikin Peak Gene have allowed prediction of the geographic location, as well as the gene location and the host animal species of each outbreak. See FIGS. 8, 9 and 10. The announcement by Dr. Krisnamurthi represents the first independent government confirmation of the success of these technological improvements. See, e.g., U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006 and U.S. application Ser. No. 11/755,597, filed May 30, 2007.
Identification of Replikin Peak Gene for Targets and Prediction
The inventors have identified a Replikin Peak Gene in an isolate of the White Spot Syndrome Virus (WSSV). A Replikin Peak Gene may be identified, isolated or synthesized for diagnosis, prevention or treatment of, for example, an outbreak of WSSV or an outbreak of a virus for which WSSV is a reservoir, such as influenza virus, by the method comprising: (1) obtaining a plurality of isolates of WSSV; (2) analyzing the protein sequences or protein sequence fragments of each isolate of the plurality of isolates for the presence and concentration of Replikin sequences; (3) identifying the protein sequence or the protein sequence fragment having the highest concentration of Replikin sequences in each isolate; (4) comparing the protein or protein fragment having the highest concentration of Replikin sequences in each isolate to the protein or protein fragment having the highest concentration of Replikin sequences in each of the other isolates of the plurality of isolates; (5) selecting the protein or protein fragment having the highest concentration of Replikin sequences; (6) identifying the amino acid sequence of the selected protein or protein fragment as the Replikin Peak Gene of the plurality of isolates of WSSV; and (7) identifying, isolating or synthesizing the identified Replikin Peak Gene of at least one of the isolates of the plurality of isolates for diagnosis, prevention or treatment. The inventors have identified a Replikin Peak Gene in a ribonucleotide reductase gene of an isolate of WSSV. See Example 1.
A Replikin Peak Gene is a segment of the genome or a protein or segment of a protein in which the expressed gene or expressed gene segment has the highest or a higher concentration of Replikins (number of Replikins per 100 amino acids) when compared to other segments or named genes of the genome. The gene or gene segment is known as the Replikin Peak Gene or Replikin Peak Gene Area. A Replikin Peak Gene has been identified in H5N1 influenza virus and an increase in concentration of Replikins in the Replikin Peak Gene of H5N1 has been correlated with epidemics, increased virulence, morbidity and human mortality. See FIGS. 11-13 and U.S. Provisional Appln. Ser. No. 60/898,097. Likewise, a Replikin Peak Gene has been identified in the VP1 protein of Foot and Mouth Disease Virus and has been correlated with outbreaks of the virus. See U.S. Provisional Appln. Ser. No. 60/954,743 (see FIG. 3 and Examples 1-4 therein). Further, a second Replikin Peak Gene has been identified in a fragment of the VP1 protein of Foot and Mouth Disease Virus and two particular Replikin sequences within the Replikin Peak Gene Area of the virus have been correlated with virulence of Foot and Mouth Disease Virus. See id. A Replikin Peak Gene has likewise been identified in West Nile Virus, Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus and Equine Influenza virus. See U.S. Provisional Appln. Ser. No. 60/853,744, filed Aug. 8, 2007 (FIG. 2 and Example 2 therein), and U.S. Provisional Appln. Ser. No. 60/935,816, filed Aug. 31, 2007 (FIGS. 1, 2 and 4).
Identification of Replikin sequences as infectious units has allowed the inventors to identify isolates having higher virulence relative to other isolates and to focus attention on the geographic area of an outbreak of virulent virus (see FIG. 10), the particular host of hosts of an outbreak of virulent virus (see FIG. 9) and the part of the genome of a virus (see FIG. 8) wherein virulence mechanisms are located and wherein increased virulence may be identified and predicted. Identification of Replikin Peak Genes within viral genomes has improved the focus that is available to make these identifications and predictions of both virulence and mechanisms of virulence.
Because the inventors have provided a method of focusing on particular units of a viral or organismal genome, the skilled artisan will understand the importance of looking for Replikin sequences in any portion of the life cycle or infectious pathway of a virus. For example, as described herein, the skilled artisan will understand that predictive and virulence-related Replikin sequences (or concentrations of Replikin sequence in Replikin Peak Genes) may be identified in reservoirs of influenza virus such as in WSSV and TSV. The skilled artisan will further understand that predictive and virulence-related Replikin sequences or Replikin Peak Genes may be identified in vectors of the influenza virus. The skilled artisan will additionally understand that predictive and virulence-related Replikin sequences or Replikin Peak Genes may be identified in hosts of the influenza virus or any other place where viral genes may be located or wherein viral genes may encounter genes of other strains of virus, other virus species, vectors or hosts.
SARS Replikin Count Correlates with Epidemics
An increase in Replikin concentration in coronaviruses also correlated with the SARS coronavirus epidemic of 2003. In particular, as may be seen in FIG. 12, Replikin concentration in Spike and Nucleocapsid Coronavirus Proteins preceded the SARS Coronavirus epidemic of 2003. In FIG. 12, the x-axis indicates the year and the y-axis indicates the Replikin concentration. The appearance of the SARS outbreak is shown by the shaded area in the graph between 2003 and 2004. The peak of the shaded area represents a total number of eight countries in which the SARS outbreak occurred in 2003. The solid black symbols represent the mean Replikin concentration for spike coronavirus proteins and the vertical black bars represent the standard deviation of the mean.
FIG. 12 shows a remarkable constancy of low coronavirus Replikin concentration between 1995 and 2001 in the spike proteins, followed by a dramatic increase in 2002, one year before the SARS epidemic appeared in 2003. Replikin concentration of the spike proteins in SARS then returned to their normal pre-2003 levels (correlated with the disappearance of SARS). The return to normal pre-2003 levels of Replikin concentration is thus a quantitative virus structural signal of the decline of the outbreak.
Replikin Concentration Correlates with Outbreaks in West Nile Virus, Foot and Mouth Disease and Equine Influenza
In all viruses observed by the inventors, significant increases in Replikin concentration have been predictive of increases in viral virulence. For example, an increase in Replikin concentration has been correlated with outbreaks in West Nile Virus, Foot and Mouth Disease, and Equine Influenza, and is predictive of outbreaks in each of these diseases.
FIG. 13 is a graph illustrating a correlation between Replikin concentration and morbidity and mortality in U.S. populations for West Nile Virus. FIG. 14 is a graph illustrating a correlation between the concentration of Replikins observed in the VP1 protein of isolates of Foot and Mouth Disease Virus between 1998 and 2006 and epidemics in the United Kingdom in 2001 and 2007.
FIG. 15 is a graph illustrating a correlation between Replikin concentration in the Replikin Peak Gene of Equine Influenza virus and epidemics between 1977 and 2000. The Replikin Peak Gene in Equine Influenza is identified as the pB1 gene area. In years where the Replikin concentration increases in the pB1 gene area of the virus, no concomitant increase in the pA or pB2 virus is observed. These data demonstrate a significant effect on virulence by an observed increase in Replikin concentration particularly in a Replikin Peak Gene.
Vaccines, Treatments and Therapeutics
The observations of specific Replikins and their concentration in WSSV and TSV proteins and their correlation with outbreaks in influenza provides for early production and timely administration of vaccines tailored specifically to treat the prevalent emerging or re-emerging strain of influenza virus in a particular region of the world. By analyzing the protein sequences of isolates of a virus for the presence, concentration and/or conservation of Replikins, virus outbreaks and epidemics can be predicted and treatments developed. Furthermore, the severity of such outbreaks can be significantly lessened by administering a peptide vaccine based on the Replikin sequences identified using the methods provided herein or Replikin sequences found to be most abundant or shown to be on the rise in virus isolates over a given time period, such as about one to about three years. Vaccine products against SARS Replikin sequences and H5N1 influenza virus Replikin Scaffolds have been demonstrated by the inventors. See, e.g., U.S. application Ser. No. 11/355,120, filed Feb. 16, 2006 (Examples 6 and 7). Replikin sequences added to the feed source of shrimp have likewise imparted measurable resistance to challenges with Taura Syndrome Virus. See Example 7 below.
A peptide vaccine may include a single Replikin peptide sequence or may include a plurality of Replikin sequences observed in particular virus strains. Preferably, the peptide vaccine is based on Replikin sequence(s) shown to be increasing in concentration over a given time period and conserved for at least that period of time. However, a vaccine may include a conserved Replikin peptide(s) in combination with a new Replikin peptide(s) or may be based on new Replikin peptide sequences. Replikin peptides can be synthesized by any method, including chemical synthesis or recombinant gene technology, and may include non-Replikin sequences, although vaccines based on peptides containing only Replikin sequences are preferred. Preferably, vaccine compositions of the invention also contain a pharmaceutically acceptable carrier and/or adjuvant.
Vaccines can be administered alone or in combination with antiviral drugs, such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.
Vaccines may be administered to any animal capable of producing antibodies in an immune response. For example, a vaccine may be administered to a rabbit, a chicken, a pig, or a human. Because of the universal nature of Replikin sequences, a vaccine of the invention may be directed at a variety of strains of virus or a particular strain of virus.
Passive Immunity
In another aspect of the invention, isolated Replikin peptides may be used to generate antibodies, which may be used, for example to provide passive immunity in an individual. Various procedures known in the art may be used for the production of antibodies to Replikin sequences. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by a Fab expression library. Antibodies that are linked to a cytotoxic agent may also be generated. Antibodies may also be administered in combination with an antiviral agent. Furthermore, combinations of antibodies to different Replikins may be administered as an antibody cocktail.
Monoclonal antibodies to Replikins may be prepared by using any technique that provides for the production of antibody molecules. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of chimeric antibodies (Morrison et al., 1984, Proc. Nat. Acad. Sci USA, 81:6851-6855) or other techniques may be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce Replikin-specific single chain antibodies.
Antibodies to any peptides observed to be present in an emerging or re-emerging strain of virus and combinations of such antibodies are useful in the treatment and/or prevention of viral infection.
Antibody fragments that contain binding sites for a Replikin may be generated by known techniques. For example, such fragments include but are not limited to F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecules and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries can be generated (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
In another aspect of the invention, immune serum containing antibodies to one or more Replikins obtained from an individual exposed to one or more Replikins may be used to induce passive immunity in another individual or animal. Immune serum may be administered via i.v. to a subject in need of treatment. Passive immunity also can be achieved by injecting a recipient with preformed antibodies to one or more Replikins. Passive immunization may be used to provide immediate protection to individuals who have been exposed to an infectious organism. Administration of immune serum or preformed antibodies is routine and the skilled practitioner can readily ascertain the amount of serum or antibodies needed to achieve the desired effect.
Replikin peptides, alone or in various combinations, may be administered to a subject, preferably by i.v. or intramuscular injection, in order to stimulate the immune system of the subject to produce antibodies to the peptide. Generally the dosage of peptides is in the range of from about 0.1 μg to about 10 mg. In another embodiment, the dosage of pepetides is about 10 μg to about 1 mg. In a preferred embodiment, the dosage of peptides is about 50 μg to about 500 μg. The skilled practitioner can readily determine the dosage and number of dosages needed to produce an effective immune response.
EXAMPLES Example 1 Determination of Very High Replikin Concentration in Ribonucleotide Reductase from Accession No. AAL89390
Replikin concentration was determined for ribonucleotide reductase publicly available at Accession No. AAL89390. The amino acid sequence was translated from the total genome of a year 2000 isolate of White Spot Syndrome Virus made publicly available at Accession No. NC 003225.1. The Replikin concentration in the protein was an unusually high 103.8 and the Replikin concentration of the Replikin Peak Gene of the protein was a yet higher 110.7. The Replikin concentration of the protein was determined by dividing the number of Replikin sequences identified in the amino acid sequence of the protein, 497 Replikin sequences, by the total amino acid length of the protein, 479 amino acids, to arrive at 103.8 Replikin sequences per 100 amino acids. The Replikin concentration of the Replikin Peak Gene was determined by dividing the number of Replikin sequences identified in the segment of the protein containing the highest concentration of Replikin sequences, 497 Replikin sequences, by the total amino acid length of the Replikin Peak Gene, 449 amino acids, to arrive at 110.7 Replikin sequences per 100 amino acids.
The amino acid sequence of the protein publicly available at Accession No. AAL89390 is of particular interest because it demonstrates an overlapping of Replikin sequences that results in very high Replikin concentrations, comparable to P. falciparum. The high concentrations of Replikin sequences provide a reservoir for transfer to influenza viruses.
In Accession No. AAL89390, the inventors identified a Replikin Peak Gene. A Replikin Peak Gene is the segment of a protein having the highest concentration of continuous, uninterrupted, overlapping, Replikin sequences. In the sequence disclosed below, the Replikin Peak Gene is underlined. The Replikin Peak Gene is observed to occupy most of the disclosed protein. The expansiveness of the Replikin Peak Gene across most of the amino acid sequence of the protein is highly unusual and creates the remarkably high Replikin concentration. The amino acid sequence further contains significant Replikin Scaffold sequences. The following Replikin Scaffold repeats were identified in Accession No. AAL89390.
(SEQ ID NO: 1)
K66KNVKSAKQLPHLKVHLDVKSAKQLPHLKVH96
(SEQ ID NO: 2)
K160KNVKSAKQLPHLKVHLDVKGVKQLLH186
(SEQ ID NO: 3)
K239KNVKSAKQLPHLKVLLDVRGAKQLPH265
(SEQ ID NO: 3)
K303KNVKSAKQLPHLKVLLDVRGAKQLPH329
(SEQ ID NO: 4)
K397KNVKSAKQLPHLKVLLDVRGAKQLPHLKVH427
Replikin Analysis for AAL89390
PubMed Code: AAL89390
Description: Ribonucleotide reductase of shrimp white spot syndrome virus (WSSV):
Isolated Year: 2000
Source: Shrimp White Spot Syndrome Virus
M1K2I3C4Q5I6S7S8P9T10L11T12L13S14I15P16L17E18G19V20Y21 H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37
V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71
A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103
Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128
V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153
K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178
K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203
V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227Q228
N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243S244A245K246Q247L248P249H250L251K252V253
L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275K276Q277L278
P279H280L281K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303
K304N305V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328
H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348L349D350V351R352G353
A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374L375K376V377L378
L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397K398N399V400K401S402A403
K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423L424K425V426H427L428
D429V430R431G432A433K434Q435Q436Q437Q438L439C440L441P442L443K444T445I446S447T448S449F450T451H452L453
L454L455C456L457Y458M459E460Y461G462K463H464Q465N466L467Q468V469K470 M471W472L473N474I475T476Y477T478
S479
(SEQ ID NO: 15)
Replikin Sequences in Amino-Terminal Portion of Peptide
(1) H22V23K24Q25L26L27H28L29K30
(SEQ ID NO: 16)
(2) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36
(SEQ ID NO: )
(3) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39
(SEQ ID NO: 18)
(4) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45
(SEQ ID NO: 19)
(5) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53
K54
(SEQ ID NO: 20)
(6) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53
K54Q55N56P57W58R59K60
(SEQ ID NO: 21)
(7) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53
K54Q55N56P57W58R59K60N61L62C63L64L65K66
(SEQ ID NO: 22)
(8) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53
K54Q55N56P57W58R59K60N61L62C63L64L65K66K67
(SEQ ID NO: 23)
(9) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53
K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70
(SEQ ID NO: 24)
(10) K24Q25L26L27H28L29K30
(SEQ ID NO: 10)
(11) K24Q25L26L27H28L29K30V31H32
(SEQ ID NO: 25)
(12) K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43
(SEQ ID NO: 26)
(13) H28L29K30V31H32L33D34V35K36
(SEQ ID NO: 27)
(14) H28L29K30V31H32L33D34V35K36G37V38K39
(SEQ ID NO: 28)
(15) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45
(SEQ ID NO: 29)
(16) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54
(SEQ ID NO: 30)
(17) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57
W58R59K60
(SEQ ID NO: 31)
(18) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57
W58R59K60N61L62C63L64L65K66
(SEQ ID NO: 32)
(19) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57
W58R59K60N61L62C63L64L65K66K67
(SEQ ID NO: 33)
(20) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57
W58R59K60N61L62C63L64L65K66K67N68V69K70
(SEQ ID NO: 34)
(21) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57
W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73
(SEQ ID NO: 35)
(22) K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59
K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 36)
(23) K30V31H32L33D34V35K36
(SEQ ID NO: 11)
(24) K30V31H32L33D34V35K36G37V38K39
(SEQ ID NO: 37)
(25) K30V31H32L33D34V35K36G37V38K39Q40L41L42H43
(SEQ ID NO: 12)
(26) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45
(SEQ ID NO: 38)
(27) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54
(SEQ ID NO: 39)
(28) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60
(SEQ ID NO: 40)
(29) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61
L62C63L64L65K66
(SEQ ID NO: 41)
(30) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61
L62C63L64L65K66K67
(SEQ ID NO: 42)
(31) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61
L62C63L64L65K66K67N68V69K70
(SEQ ID NO: 43)
(32) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61
L62C63L64L65K66K67N68V69K70S71A72K73
(SEQ ID NO: 44)
(33) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61
L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79
(SEQ ID NO: 45)
(34) K36G37V38K39Q40L41L42H43L44K45
(SEQ ID NO: 46)
(35) K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65
K66K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 47)
(36) K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65
K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 48)
(37) K39Q40L41L42H43L44K45
(SEQ ID NO: 10)
(38) K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68
V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 49)
(39) K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68
V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 50)
(40) H43L44K45V46R47L48D49V50R51G52A53K54
(SEQ ID NO: 51)
(41) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60
(SEQ ID NO: 52)
(42) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66
(SEQ ID NO: 53)
(43) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67
(SEQ ID NO: 54)
(44) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70
(SEQ ID NO: 55)
(45) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72
K73
(SEQ ID NO: 56)
(46) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72
K73Q74L75P76H77L78K79
(SEQ ID NO: 57)
(47) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72
K73Q74L75P76H77L78K79V80H81L82D83V84K85
(SEQ ID NO: 58)
(48) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72
K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88
(SEQ ID NO: 59)
(49) K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74
L75P76H77
(SEQ ID NO: 60)
(50) K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74
L75P76H77L78K79V80H81
(SEQ ID NO: 61)
(51) K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74
L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 62)
(52) K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 63)
(53) K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 64)
(54) K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85
S86A87K88Q89L90P91H92
(SEQ ID NO: 65)
(55) K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85
S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 66)
(56) K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91
H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 67)
(57) K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 68)
(58) K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 69)
(59) K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91
H92
(SEQ ID NO: 70)
(60) K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91
H92L93K94V95H96
(SEQ ID NO: 71)
(61) K66K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 13)
(62) K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 72)
(63) K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 73)
(64) K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 1)
(65) K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97
D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 74)
(66) K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 75)
(67) K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 76)
(68) K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 77)
(69) K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 78)
(70) K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98
V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 79)
(71) K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100
G101A102K103Q104L105P106H107
(SEQ ID NO: 80)
(72) K70S71A72K73Q74L75P76H77L78K79
(SEQ ID NO: 14)
(73) K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 81)
(74) K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 82)
(75) K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 83)
(76) K73Q74L75P76H77L78K79
(SEQ ID NO: 84)
(77) K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 85)
(78) K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102
K103Q104L105P106H107
(SEQ ID NO: 86)
(79) K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 87)
(80) K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 88)
(81) K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102
K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122
(SEQ ID NO: 89)
(82) H77L78K79V80H81L82D83V84K85
(SEQ ID NO: 27)
(83) H77L78K79V80H81L82D83V84K85S86A87K88
(SEQ ID NO: 90)
(84) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94
(SEQ ID NO: 91)
(85) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103
(SEQ ID NO: 92)
(86) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105
P106H107L108K109
(SEQ ID NO: 93)
(87) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105
P106H107L108K109V110R111L112D113V114K115
(SEQ ID NO: 94)
(88) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105
P106H107L108K109V110R111L112D113V114K115S116A117K118
(SEQ ID NO: 95)
(89) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105
P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124
(SEQ ID NO: 96)
(90) K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 97)
(91) K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122
(SEQ ID NO: 98)
(92) K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 99)
(93) K79V80H81L82D83V84K85
(SEQ ID NO: 11)
(94) K79V80H81L82D83V84K85S86A87K88
(SEQ ID NO: 100)
(95) K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 101)
(96) K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 102)
(97) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94
(SEQ ID NO: 103)
(98) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103
(SEQ ID NO: 104)
(99) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108
K109
(SEQ ID NO: 105)
(100) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108
K109V110R111L112D113V114K115
(SEQ ID NO: 106)
(101) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108
K109V110R111L112D113V114K115S116A117K118
(SEQ ID NO: 107)
(102) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108
K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124
(SEQ ID NO: 108)
(103) K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 109)
(104) K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111
L112D113V114K115S116A117K118Q119L120P121H122
(SEQ ID NO: 110)
(105) K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111
L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 111)
(106) K85S86A87K88Q89L90P91H92L93K94
(SEQ ID NO: 14)
(107) K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 82)
(108) K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 112)
(109) K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114
K115S116A117K118Q119L120P121H122
(SEQ ID NO: 113)
(110) K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114
K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 114)
(111) K88Q89L90P91H92L93K94
(SEQ ID NO: 84)
(112) K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 85)
(113) K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114
K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 115)
(114) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115
(SEQ ID NO: 116)
(115) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116
A117K118
(SEQ ID NO: 117)
(116) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116
A117K118Q119L120P121H122L123K124
(SEQ ID NO: 118)
(117) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116
A117K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133
(SEQ ID NO: 119)
(118) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116
A117K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139
(SEQ ID NO: 120)
(119) H92L93K94V95H96L97D98V99R100G101A102K103
(SEQ ID NO: 121)
(120) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109
(SEQ ID NO: 122)
(121) K94V95H96L97D98V99R100G101A102K103
(SEQ ID NO: 123)
(122) K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 124)
(123) K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118
Q119L120P121H122
(SEQ ID NO: 125)
(124) K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118
Q119L120P121H122L123K124V125H126
(SEQ ID NO: 126)
(125) K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118
Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 127)
(126) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109
(SEQ ID NO: 128)
(127) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115
(SEQ ID NO: 129)
(128) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118
(SEQ ID NO: 130)
(129) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120
P121H122L123K124
(SEQ ID NO: 131)
(130) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120
P121H122L123K124V125H126L127D128V129R130G131A132K133
(SEQ ID NO: 132)
(131) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120
P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139
(SEQ ID NO: 133)
(132) K103Q104L105P106H107L108K109
(SEQ ID NO: 84)
(133) K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122
(SEQ ID NO: 134)
(134) K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 135)
(135) K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126
L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 136)
(136) H107L108K109V110R111L112D113V114K115
(SEQ ID NO: 137)
(137) H107L108K109V110R111L112D113V114K115S116A117K118
(SEQ ID NO: 138)
(138) H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124
(SEQ ID NO: 139)
(139) H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130
G131A132K133
(SEQ ID NO: 140)
(140) H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130
G131A132K133Q134L135P136H137L138K139
(SEQ ID NO: 141)
(141) H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130
G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148
(SEQ ID NO: 142)
(142) H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130
G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154
(SEQ ID NO: 143)
(143) K109V110R111L112D113V114K115S116A117K118Q119L120P121H122
(SEQ ID NO: 144)
(144) K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 145)
(145) K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132
K133Q134L135P136H137
(SEQ ID NO: 146)
(146) K115S116A117K118Q119L120P121H122L123K124
(SEQ ID NO: 14)
(147) K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 81)
(148) K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 109)
(149) K118Q119L120P121H122L123K124
(SEQ ID NO: 84)
(150) K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 85)
(151) K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 112)
(152) H122L123K124V125H126L127D128V129R130G131A132K133
(SEQ ID NO: 121)
(153) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139
(SEQ ID NO: 122)
(154) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148
(SEQ ID NO: 147)
(155) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148Q149N150P151W152R153K154
(SEQ ID NO: 148)
(156) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
(SEQ ID NO: 149)
(157) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161
(SEQ ID NO: 150)
(158) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164
(SEQ ID NO: 151)
(159) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167
(SEQ ID NO: 152)
(160) K124V125H126L127D128V129R130G131A132K133
(SEQ ID NO: 123)
(161) K124V125H126L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 124)
(162) K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147
K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 153)
(163) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139
(SEQ ID NO: 128)
(164) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148
(SEQ ID NO: 154)
(165) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154
(SEQ ID NO: 155)
(166) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154N155L156C157L158L159K160
(SEQ ID NO: 156)
(167) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154N155L156C157L158L159K160K161
(SEQ ID NO: 157)
(168) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164
(SEQ ID NO: 158)
(169) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167
(SEQ ID NO: 159)
(170) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173
(SEQ ID NO: 160)
(171) K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156
C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 161)
(172) K133Q134L135P136H137L138K139
(SEQ ID NO: 84)
(173) K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156
C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 162)
(174) H137L138K139V140R141L142D143V144R145G146A147K148
(SEQ ID NO: 51)
(175) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154
(SEQ ID NO: 52)
(176) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
(SEQ ID NO: 53)
(177) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161
(SEQ ID NO: 54)
(178) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161N162V163K164
(SEQ ID NO: 55)
(179) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161N162V163K164S165A166K167
(SEQ ID NO: 56)
(180) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161N162V163K164S165A166K167Q168L169P170H171L172K173
(SEQ ID NO: 57)
(181) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179
(SEQ ID NO: 58)
(182) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182
(SEQ ID NO: 163)
(183) K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162
V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 60)
(184) K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162
V163K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 61)
(185) K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162
V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 164)
(186) K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 63)
(187) K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
L172K173V174H175
(SEQ ID NO: 64)
(188) K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 165)
(189) K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177
V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201
(SEQ ID NO: 166)
(190) K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 68)
(191) K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 69)
(192) K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177
V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 167)
(193) K160K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 13)
(194) K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 72)
(195) K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183
L184L185H186
(SEQ ID NO: 2)
(196) K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183
L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201
(SEQ ID NO: 168)
(197) K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183
L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205
(SEQ ID NO: 169)
Replikin Sequences in Mid-molecule Portion of Peptide
(198) K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 75)
(199) K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 76)
(200) K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184
L185H186
(SEQ ID NO: 170)
(201) K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184
L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201
(SEQ ID NO: 171)
(202) K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184
L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205
(SEQ ID NO: 172)
(203) K164S165A166K167Q168L169P170H171L172K173
(SEQ ID NO: 14)
(204) K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 81)
(205) K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 173)
(206) K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187
K188V189R190L191D192V193R194G195A196K197Q198L199L200H201
(SEQ ID NO: 174)
(207) K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187
K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205
(SEQ ID NO: 175)
(208) K167Q168L169P170H171L172K173
(SEQ ID NO: 84)
(209) K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 85)
(210) K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 176)
(211) K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190
L191D192V193R194G195A196K197Q198L199L200H201
(SEQ ID NO: 177)
(212) K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190
L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205
(SEQ ID NO: 178)
(213) K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190
L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205L206D207V208R209G210A211K212Q213L214
P215H216
(SEQ ID NO: 179)
(214) H171L172K173V174H175L176D177V178K179
(SEQ ID NO: 27)
(215) H171L172K173V174H175L176D177V178K179G180V181K182
(SEQ ID NO: 28)
(216) H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188
(SEQ ID NO: 29)
(217) H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194
G195A196K197Q198L199L200H201L202K203
(SEQ ID NO: 180)
(218) H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194
G195A196K197
(SEQ ID NO: 30)
(219) H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194
G195A196K197Q198L199L200H201L202K203V204H205L206D207V208R209G210A211K212
(SEQ ID NO: 181)
(220) H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194
G195A196K197Q198L199L200H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218
(SEQ ID NO: 182)
(221) K173V174H175L176D177V178K179
(SEQ ID NO: 11)
(222) K173V174H175L176D177V178K179G180V181K182
(SEQ ID NO: 37)
(223) K173V174H175L176D177V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 12)
(224) K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196
K197Q198L199L200H201
(SEQ ID NO: 183)
(225) K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196
K197Q198L199L200H201L202K203V204H205
(SEQ ID NO: 184)
(226) K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196
K197Q198L199L200H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216
(SEQ ID NO: 185)
(227) K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196
K197Q198L199L200H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220
(SEQ ID NO: 186)
(228) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198
L199L200H201L202K203V204H205L206D207V208R209G210A211K212
(SEQ ID NO: 187)
(229) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198
L199L200H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218
(SEQ ID NO: 188)
(230) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188
(SEQ ID NO: 38)
(231) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198
L199L200H201L202K203
(SEQ ID NO: 189)
(232) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197
(SEQ ID NO: 39)
(233) K179G180V181K182Q183L184L185H186L187K188
(SEQ ID NO: 46)
(234) K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201
(SEQ ID NO: 190)
(235) K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202
K203V204H205
(SEQ ID NO: 191)
(236) K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202
K203V204H205L206D207V208R209G210A211K212Q213L214P215H216
(SEQ ID NO: 192)
(237) K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202
K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220
(SEQ ID NO: 193)
(238) K182Q183L184L185H186L187K188
(SEQ ID NO: 10)
(239) K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201
(SEQ ID NO: 194)
(240) K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205
(SEQ ID NO: 195)
(241) K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205
L206D207V208R209G210A211K212Q213L214P215H216
(SEQ ID NO: 196)
(242) K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205
L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220
(SEQ ID NO: 197)
(243) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203
(SEQ ID NO: 198)
(244) H186L187K188V189R190L191D192V193R194G195A196K197
(SEQ ID NO: 51)
(245) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205L206D207V208R209
G210A211K212
(SEQ ID NO: 199)
(246) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205L206D207V208R209
G210A211K212Q213L214P215H216L217K218
(SEQ ID NO: 200)
(247) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205L206D207V208R209
G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227
(SEQ ID NO: 201)
(248) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205L206D207V208R209
G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233
(SEQ ID NO: 202)
(249) K188V189R190L191D192V193R194G195A196K197Q198L199L200H201
(SEQ ID NO: 203)
(250) K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205
(SEQ ID NO: 204)
(251) K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205L206D207V208R209G210A211
K212Q213L214P215H216
(SEQ ID NO: 205)
(252) K188V189R190L191D192V193R194G195A196K197Q198L199L200H201L202K203V204H205L206D207V208R209G210A211
K212Q213L214P215H216L217K218V219H220
(SEQ ID NO: 206)
(253) K197Q198L199L200H201L202K203
(SEQ ID NO: 10)
(254) K197Q198L199L200H201L202K203V204H205
(SEQ ID NO: 25)
(255) K197Q198L199L200H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216
(SEQ ID NO: 207)
(256) K197Q198L199L200H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220
(SEQ ID NO: 208)
(257) H201L202K203V204H205L206D207V208R209G210A2111K212
(SEQ ID NO: 121)
(258) H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218
(SEQ ID NO: 122)
(259) H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224
G225A226K227
(SEQ ID NO: 209)
(260) H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224
G225A226K227Q228N229P230W231R232K233
(SEQ ID NO: 210)
(261) H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224
G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239
(SEQ ID NO: 211)
(262) H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224
G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240
(SEQ ID NO: 212)
(263) H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224
G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243
(SEQ ID NO: 213)
(264) H201L202K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224
G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243S244A245K246
(SEQ ID NO: 214)
(265) K203V204H205L206D207V208R209G210A211K212
(SEQ ID NO: 123)
(266) K203V204H205L206D207V208R209G210A211K212Q213L214P215H216
(SEQ ID NO: 124)
(267) K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220
(SEQ ID NO: 215)
(268) K203V204H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226
K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243S244A245K246Q247L248P249H250
(SEQ ID NO: 216)
(269) H205L206D207V208R209G210A211K212Q213L214P215H216L217K218
(SEQ ID NO: 128)
(270) H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227
(SEQ ID NO: 217)
(271) H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227Q228
N229P230W231R232K233
(SEQ ID NO: 218)
(272) H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227Q228
N229P230W231R232K233N234L235C236L237L238K239
(SEQ ID NO: 219)
(273) H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227Q228
N229P230W231R232K233N234L235C236L237L238K239K240
(SEQ ID NO: 220)
(274) H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227Q228
N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243
(SEQ ID NO: 221)
(275) H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227Q228
N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243S244A245K246
(SEQ ID NO: 222)
(276) H205L206D207V208R209G210A211K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227Q228
N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243S244A245K246Q247L248P249H250L251K252
(SEQ ID NO: 223)
(277) K212Q213L214P215H216L217K218
(SEQ ID NO: 84)
(278) K212Q213L214P215H216L217K218V219H220
(SEQ ID NO: 85)
(279) K212Q213L214P215H216L217K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235
C236L237L238K239K240N241V242K243S244A245K246Q247L248P249H250
(SEQ ID NO: 224)
(280) H216L217K218V219H220L221D222V223R224G225A226K227
(SEQ ID NO: 121)
(281) H216L217K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233
(SEQ ID NO: 225)
(282) H216L217K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239
(SEQ ID NO: 226)
(283) H216L217K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239
K240
(SEQ ID NO: 227)
(284) H216L217K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239
K240N241V242K243
(SEQ ID NO: 228)
(285) H216L217K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239
K240N241V242K243S244A245K246
(SEQ ID NO: 229)
(286) H216L217K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239
K240N241V242K243S244A245K246Q247L248P249H250L251K252
(SEQ ID NO: 230)
(287) H216L217K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239
K240N241V242K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261
(SEQ ID NO: 231)
(288) K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241
V242K243S244A245K246Q247L248P249H250
(SEQ ID NO: 232)
(289) K218V219H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241
V242K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 233)
(290) K218V219H220L221D222V223R224G225A226K227
(SEQ ID NO: 123)
(291) H220L221D222V223R224G225A226K227Q228N229P230W231R232K233
(SEQ ID NO: 234)
(292) H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239
(SEQ ID NO: 235)
(293) H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240
(SEQ ID NO: 236)
(294) H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243
(SEQ ID NO: 237)
(295) H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243
S244A245K246
(SEQ ID NO: 238)
(296) H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243
S244A245K246Q247L248P249H250L251K252
(SEQ ID NO: 239)
(297) H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243
S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261
(SEQ ID NO: 240)
(298) H220L221D222V223R224G225A226K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243
S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267
(SEQ ID NO: 241)
(299) K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243S244A245K246Q247L248P249H250
(SEQ ID NO: 63)
(300) K227Q228N229P230W231R232K233N234L235C236L237L238K239K240N241V242K243S244A245K246Q247L248P249H250
L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 242)
(301) K233N234L235C236L237L238K239K240N241V242K243S244A245K246Q247L248P249H250
(SEQ ID NO: 68)
(302) K233N234L235C236L237L238K239K240N241V242K243S244A245K246Q247L248P249H250L251K252V253L254L255D256
V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 243)
(303) K233N234L235C236L237L238K239K240N241V242K243S244A245K246Q247L248P249H250L251K252V253L254L255D256
V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280
(SEQ ID NO: 244)
(304) K239K240N241V242K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262
L263P264H265
(SEQ ID NO: 3)
(305) K239K240N241V242K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262
L263P264H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280
(SEQ ID NO: 245)
(306) K239K240N241V242K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262
L263P264H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284
(SEQ ID NO: 246)
(307) K239K240N241V242K243S244A245K246Q247L248P249H250
(SEQ ID NO: 13)
(308) K240N241V242K243S244A245K246Q247L248P249H250
(SEQ ID NO: 75)
(309) K240N241V242K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263
P264H265
(SEQ ID NO: 247)
(310) K240N241V242K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263
P264H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280
(SEQ ID NO: 248)
(311) K240N241V242K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263
P264H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284
(SEQ ID NO: 249)
(312) K243S244A245K246Q247L248P249H250L251K252
(SEQ ID NO: 14)
(313) K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 250)
(314) K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266
K267V268L269L270D271V272R273G274A275K276Q277L278P279H280
(SEQ ID NO: 251)
(315) K243S244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266
K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284
(SEQ ID NO: 252)
(316) K246Q247L248P249H250L251K252
(SEQ ID NO: 84)
(317) K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 253)
(318) K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269
L270D271V272R273G274A275K276Q277L278P279H280
(SEQ ID NO: 254)
(319) K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269
L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284
(SEQ ID NO: 255)
(320) H250L251K252V253L254L255D256V257R258G259A260K261
(SEQ ID NO: 256)
(321) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267
(SEQ ID NO: 257)
(322) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273
G274A275K276
(SEQ ID NO: 258)
(323) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273
G274A275K276Q277L278P279H280L281K282
(SEQ ID NO: 259)
(324) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273
G274A275K276Q277L278P279H280L281K282V283H284L285D286V287R288G289A290K291
(SEQ ID NO: 260)
(325) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273
G274A275K276Q277L278P279H280L281K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297
(SEQ ID NO: 261)
(326) K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 262)
(327) K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275
K276Q277L278P279H280
(SEQ ID NO: 263)
(328) K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275
K276Q277L278P279H280L281K282V283H284
(SEQ ID NO: 264)
(329) K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280
(SEQ ID NO: 253)
(330) K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284
(SEQ ID NO: 265)
(331) K261Q262L263P264H265L266K267
(SEQ ID NO: 84)
(332) H265L266K267V268L269L270D271V272R273G274A275K276
(SEQ ID NO: 256)
(333) H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282
(SEQ ID NO: 257)
(334) H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284L285D286V287R288
G289A290K291
(SEQ ID NO: 266)
(335) H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284L285D286V287R288
G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303
(SEQ ID NO: 267)
(336) H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284L285D286V287R288
G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303K304
(SEQ ID NO: 268)
(337) H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284L285D286V287R288
G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305V306K307
(SEQ ID NO: 269)
(338) H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284L285D286V287R288
G289A290K291Q292N293P294W295R296K297
(SEQ ID NO: 270)
(339) H265L266K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284L285D286V287R288
K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305V306K307S308A309K310
(SEQ ID NO: 271)
(340) K267V268L269L270D271V272R273G274A275K276Q277L278P279H280
(SEQ ID NO: 264)
(341) K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284
(SEQ ID NO: 272)
(342) K267V268L269L270D271V272R273G274A275K276Q277L278P279H280L281K282V283H284L285D286V287R288G289A290
K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305V306K307S308A309K310Q311L312P313H314
(SEQ ID NO: 273)
(343) K276Q277L278P279H280L281K282
(SEQ ID NO: 84)
(344) K276Q277L278P279H280L281K282V283H284
(SEQ ID NO: 85)
(345) K276Q277L278P279H280L281K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299
C300L301L302K303K304N305V306K307S308A309K310Q311L312P313H314
(SEQ ID NO: 224)
(346) H280L281K282V283H284L285D286V287R288G289A290K291
(SEQ ID NO: 121)
(347) H280L281K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303
(SEQ ID NO: 226)
(348) H280L281K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303
K304
(SEQ ID NO: 227)
(349) H280L281K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303
K304N305V306K307
(SEQ ID NO: 228)
(350) H280L281K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297
(SEQ ID NO: 225)
(351) H280L281K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303
K304N305V306K307S308A309K310
(SEQ ID NO: 229)
(352) H280L281K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303
K304N305V306K307S308A309K310Q311L312P313H314L315K316
(SEQ ID NO: 230)
(353) H280L281K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303
K304N305V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325
(SEQ ID NO: 231)
(354) K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305
V306K307S308A309K310Q311L312P313H314
(SEQ ID NO: 232)
(355) K282V283H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305
V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329
(SEQ ID NO: 233)
(356) K282V283H284L285D286V287R288G289A290K291
(SEQ ID NO: 123)
(357) H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303
(SEQ ID NO: 235)
(358) H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303K304
(SEQ ID NO: 236)
(359) H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305V306K307
(SEQ ID NO: 237)
(360) H284L285D286V287R288G289A290K291Q292N293P294W295R296K297
(SEQ ID NO: 234)
(361) H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305V306K307
S308A309K310
(SEQ ID NO: 238)
(362) H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305V306K307
S308A309K310Q311L312P313H314L315K316
(SEQ ID NO: 239)
(363) H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305V306K307
S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325
(SEQ ID NO: 240)
(364) H284L285D286V287R288G289A290K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305V306K307
S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331
(SEQ ID NO: 241)
(365) K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305V306K307S308A309K310Q311L312P313H314
(SEQ ID NO: 63)
(366) K291Q292N293P294W295R296K297N298L299C300L301L302K303K304N305V306K307S308A309K310Q311L312P313H314
L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329
(SEQ ID NO: 242)
(367) K297N298L299C300L301L302K303K304N305V306K307S308A309K310Q311L312P313H314
(SEQ ID NO: 68)
(368) K297N298L299C300L301L302K303K304N305V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320
V321R322G323A324K325Q326L327P328H329
(SEQ ID NO: 243)
(369) K297N298L299C300L301L302K303K304N305V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320
V321R322G323A324K325Q326L327P328H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344
(SEQ ID NO: 244)
(370) K303K304N305V306K307S308A309K310Q311L312P313H314
(SEQ ID NO: 13)
(371) K303K304N305V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326
L327P328H329
(SEQ ID NO: 3)
(372) K303K304N305V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326
L327P328H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344
(SEQ ID NO: 245)
(373) K303K304N305V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326
L327P328H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348
(SEQ ID NO: 276)
(374) K304N305V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327
P328H329
(SEQ ID NO: 247)
(375) K304N305V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327
P328H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344
(SEQ ID NO: 248)
(376) K304N305V306K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327
P328H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348
(SEQ ID NO: 249)
(377) K304N305V306K307S308A309K310Q311L312P313H314
(SEQ ID NO: 75)
(378) K307S308A309K310Q311L312P313H314L315K316
(SEQ ID NO: 14)
(379) K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329
(SEQ ID NO: 250)
(380) K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330
K331V332L333L334D335V336R337G338A339K340Q341L342P343H344
(SEQ ID NO: 251)
(381) K307S308A309K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330
K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348
(SEQ ID NO: 252)
(382) K310Q311L312P313H314L315K316
(SEQ ID NO: 84)
(383) K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329
(SEQ ID NO: 253)
(384) K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331V332L333
L334D335V336R337G338A339K340Q341L342P343H344
(SEQ ID NO: 254)
(385) K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331V332L333
L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348
(SEQ ID NO: 255)
(386) K310Q311L312P313H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331V332L333
L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348L349D350V351R352G353A354K355Q356L357
P358H359
(SEQ ID NO: 277)
(387) H314L315K316V317L318L319D320V321R322G323A324K325
(SEQ ID NO: 256)
(388) H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331
(SEQ ID NO: 257)
(389) H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331V332L333L334D335V336R337
G338A339K340
(SEQ ID NO: 258)
(390) H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331V332L333L334D335V336R337
G338A339K340Q341L342P343H344L345K346
(SEQ ID NO: 259)
(391) H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331V332L333L334D335V336R337
G338A339K340Q341L342P343H344L345K346V347H348L349D350V351R352G353A354K355
(SEQ ID NO: 260)
(392) H314L315K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331V332L333L334D335V336R337
G338A339K340Q341L342P343H344L345K346V347H348L349D350V351R352G353A354K355Q356L357P358H359L360K361
(SEQ ID NO: 278)
(393) K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331V332L333L334D335V336R337G338A339
K340Q341L342P343H344L345K346V347H348L349D350V351R352G353A354K355Q356L357P358H359
(SEQ ID NO: 279)
(394) K316V317L318L319D320V321R322G323A324K325Q326L327P328H329
(SEQ ID NO: 262)
(395) K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331V332L333L334D335V336R337G338A339
K340Q341L342P343H344
(SEQ ID NO: 263)
(396) K316V317L318L319D320V321R322G323A324K325Q326L327P328H329L330K331V332L333L334D335V336R337G338A339
K340Q341L342P343H344L345K346V347H348
(SEQ ID NO: 264)
Replikin Sequences in Carboxy-Terminal Portion of Peptide
(397) K325Q326L327P328H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344
(SEQ ID NO: 253)
(398) K325Q326L327P328H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348
(SEQ ID NO: 265)
(399) K325Q326L327P328H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348
L349D350V351R352G353A354K355Q356L357P358H359
(SEQ ID NO: 280)
(400) K325Q326L327P328H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348
L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370Q371L372
P373H374
(SEQ ID NO: 281)
(401) K325Q326L327P328H329L330K331
(SEQ ID NO: 84)
(402) H329L330K331V332L333L334D335V336R337G338A339K340
(SEQ ID NO: 256)
(403) H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346
(SEQ ID NO: 257)
(404) H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348L349D350V351R352
G353A354K355
(SEQ ID NO: 266)
(405) H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348L349D350V351R352
G353A354K355Q356L357P358H359L360K361
(SEQ ID NO: 282)
(406) H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348L349D350V351R352
G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370
(SEQ ID NO: 283)
(407) H329L330K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348L349D350V351R352
G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374L375K376
(SEQ ID NO: 284)
(408) K331V332L333L334D335V336R337G338A339K340Q341L342P343H344
(SEQ ID NO: 262)
(409) K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348
(SEQ ID NO: 272)
(410) K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348L349D350V351R352G353A354
K355Q356L357P358H359
(SEQ ID NO: 285)
(411) K331V332L333L334D335V336R337G338A339K340Q341L342P343H344L345K346V347H348L349D350V351R352G353A354
K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374
(SEQ ID NO: 286)
(412) K340Q341L342P343H344L345K346
(SEQ ID NO: 84)
(413) K340Q341L342P343H344L345K346V347H348
(SEQ ID NO: 85)
(414) K340Q341L342P343H344L345K346V347H348L349D350V351R352G353A354K355Q356L357P358H359
(SEQ ID NO: 112)
(415) K340Q341L342P343H344L345K346V347H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363
L364D365V366R367G368A369K370Q371L372P373H374
(SEQ ID NO: 287)
(416) H344L345K346V347H348L349D350V351R352G353A354K355
(SEQ ID NO: 121)
(417) H344L345K346V347H348L349D350V351R352G353A354K355Q356L357P358H359L360K361
(SEQ ID NO: 122)
(418) H344L345K346V347H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367
G368A369K370
(SEQ ID NO: 288)
(419) H344L345K346V347H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367
G368A369K370Q371L372P373H374L375K376
(SEQ ID NO: 289)
(420) H344L345K346V347H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367
G368A369K370Q371L372P373H374L375K376V377L378L379D380V381R382G383A384K385
(SEQ ID NO: 290)
(421) H344L345K346V347H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367
G368A369K370Q371L372P373H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391
(SEQ ID NO: 291)
(422) K346V347H348L349D350V351R352G353A354K355
(SEQ ID NO: 123)
(423) K346V347H348L349D350V351R352G353A354K355Q356L357P358H359
(SEQ ID NO: 124)
(424) K346V347H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369
K370Q371L372P373H374
(SEQ ID NO: 292)
(425) H348L349D350V351R352G353A354K355Q356L357P358H359L360K361
(SEQ ID NO: 128)
(426) H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370
(SEQ ID NO: 293)
(427) H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370Q371
L372P373H374L375K376
(SEQ ID NO: 294)
(428) H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370Q371
L372P373H374L375K376V377L378L379D380V381R382G383A384K385
(SEQ ID NO: 295)
(429) H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370Q371
L372P373H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391
(SEQ ID NO: 296)
(430) H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370Q371
L372P373H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395
L396K397
(SEQ ID NO: 297)
(431) H348L349D350V351R352G353A354K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370Q371
L372P373H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395
L396K397K398
(SEQ ID NO: 298)
(432) K355Q356L357P358H359L360K361
(SEQ ID NO: 84)
(433) K355Q356L357P358H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374
(SEQ ID NO: 253)
(434) H359L360K361V362L363L364D365V366R367G368A369K370
(SEQ ID NO: 256)
(435) H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374L375K376
(SEQ ID NO: 257)
(436) H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374L375K376V377L378L379D380V381R382
G383A384K385
(SEQ ID NO: 258)
(437) H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374L375K376V377L378L379D380V381R382
G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397K398N399V400K401
(SEQ ID NO: 299)
(438) H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374L375K376V377L378L379D380V381R382
G383A384K385Q386N387P388W389R390K391
(SEQ ID NO: 300)
(439) H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374L375K376V377L378L379D380V381R382
G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397K398N399V400K401S402A403K404
(SEQ ID NO: 301)
(440) H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374L375K376V377L378L379D380V381R382
G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397
(SEQ ID NO: 302)
(441) H359L360K361V362L363L364D365V366R367G368A369K370Q371L372P373H374L375K376V377L378L379D380V381R382
G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397K398
(SEQ ID NO: 303)
(442) K361V362L363L364D365V366R367G368A369K370Q371L372P373H374
(SEQ ID NO: 262)
(443) K361V362L363L364D365V366R367G368A369K370Q371L372P373H374L375K376V377L378L379D380V381R382G383A384
K385Q386N387P388W389R390K391N392L393C394L395L396K397K398N399V400K401S402A403K404Q405L406P407H408
(SEQ ID NO: 304)
(444) K370Q371L372P373H374L375K376
(SEQ ID NO: 84)
(445) K370Q371L372P373H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393
C394L395L396K397K398N399V400K401S402A403K404Q405L406P407H408
(SEQ ID NO: 305)
(446) H374L375K376V377L378L379D380V381R382G383A384K385
(SEQ ID NO: 256)
(447) H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397
K398N399V400K401
(SEQ ID NO: 306)
(448) H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391
(SEQ ID NO: 307)
(449) H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397
K398N399V400K401S402A403K404
(SEQ ID NO: 308)
(450) H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397
(SEQ ID NO: 309)
(451) H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397
K398
(SEQ ID NO: 310)
(452) H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397
K398N399V400K401S402A403K404Q405L406P407H408L409K410
(SEQ ID NO: 311)
(453) H374L375K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397
K398N399V400K401S402A403K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419
(SEQ ID NO: 312)
(454) K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397K398N399
V400K401S402A403K404Q405L406P407H408
(SEQ ID NO: 313)
(455) K376V377L378L379D380V381R382G383A384K385Q386N387P388W389R390K391N392L393C394L395L396K397K398N399
V400K401S402A403K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423
(SEQ ID NO: 314)
(456) K385Q386N387P388W389R390K391N392L393C394L395L396K397K398N399V400K401S402A403K404Q405L406P407H408
(SEQ ID NO: 63)
(457) K385Q386N387P388W389R390K391N392L393C394L395L396K397K398N399V400K401S402A403K404Q405L406P407H408
L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423
(SEQ ID NO: 242)
(458) K385Q386N387P388W389R390K391N392L393C394L395L396K397K398N399V400K401S402A403K404Q405L406P407H408
L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423L424K425V426H427
(SEQ ID NO: 315)
(459) K391N392L393C394L395L396K397K398N399V400K401S402A403K404Q405L406P407H408
(SEQ ID NO: 68)
(460) K391N392L393C394L395L396K397K398N399V400K401S402A403K404Q405L406P407H408L409K410V411L412L413D414
V415R416G417A418K419Q420L421P422H423
(SEQ ID NO: 243)
(461) K391N392L393C394L395L396K397K398N399V400K401S402A403K404Q405L406P407H408L409K410V411L412L413D414
V415R416G417A418K419Q420L421P422H423L424K425V426H427
(SEQ ID NO: 316)
(462) K397K398N399V400K401S402A403K404Q405L406P407H408
(SEQ ID NO: 13)
(463) K397K398N399V400K401S402A403K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420
L421P422H423
(SEQ ID NO: 3)
(464) K397K398N399V400K401S402A403K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420
L421P422H423L424K425V426H427
(SEQ ID NO: 4)
(465) K398N399V400K401S402A403K404Q405L406P407H408
(SEQ ID NO: 75)
(466) K398N399V400K401S402A403K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420L421
P422H423
(SEQ ID NO: 247)
(467) K398N399V400K401S402A403K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420L421
P422H423L424K425V426H427
(SEQ ID NO: 318)
(468) K401S402A403K404Q405L406P407H408L409K410
(SEQ ID NO: 14)
(469) K401S402A403K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423
(SEQ ID NO: 250)
(470) K401S402A403K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423L424
K425V426H427
(SEQ ID NO: 319)
(471) K404Q405L406P407H408L409K410
(SEQ ID NO: 84)
(472) K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423
(SEQ ID NO: 253)
(473) K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423L424K425V426H427
(SEQ ID NO: 265)
(474) K404Q405L406P407H408L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423L424K425V426H427
L428D429V430R431G432A433K434Q435Q436Q437Q438L439C440L441P442L443K444T445I446S447T448S449F450T451
H452
(SEQ ID NO: 320)
(475) H408L409K410V411L412L413D414V415R416G417A418K419
(SEQ ID NO: 256)
(476) H408L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423L424K425
(SEQ ID NO: 257)
(477) H408L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423L424K425V426H427L428D429V430R431
G432A433K434
(SEQ ID NO: 266)
(478) H408L409K410V411L412L413D414V415R416G417A418K419Q420L421P422H423L424K425V426H427L428D429V430R431
G432A433K434Q435Q436Q437Q438L439C440L441P442L443K444
(SEQ ID NO: 321)
(479) K410V411L412L413D414V415R416G417A418K419Q420L421P422H423
(SEQ ID NO: 262)
(480) K410V411L412L413D414V415R416G417A418K419Q420L421P422H423L424K425V426H427
(SEQ ID NO: 272)
(481) K410V411L412L413D414V415R416G417A418K419Q420L421P422H423L424K425V426H427L428D429V430R431G432A433
K434Q435Q436Q437Q438L439C440L441P442L443K444T445I446S447T448S449F450T451H452
(SEQ ID NO: 322)
(482) K419Q420L421P422H423L424K425
(SEQ ID NO: 84)
(483) K419Q420L421P422H423L424K425V426H427
(SEQ ID NO: 85)
(484) K419Q420L421P422H423L424K425V426H427L428D429V430R431G432A433K434Q435Q436Q437Q438L439C440L441P442
L443K444T445I446S447T448S449F450T451H452
(SEQ ID NO: 323)
(485) K419Q420L421P422H423L424K425V426H427L428D429V430R431G432A433K434Q435Q436Q437Q438L439C440L441P442
L443K444T445I446S447T448S449F450T451H452L453L454L455C456L457Y458M459E460Y461G462K463H464
(SEQ ID NO: 324)
(486) H423L424K425V426H427L428D429V430R431G432A433K434Q435Q436Q437Q438L439C440L441P442L443K444
(SEQ ID NO: 325)
(487) H423L424K425V426H427L428D429V430R431G432A433K434Q435Q436Q437Q438L439C440L441P442L443K444T445I446
S447T448S449F450T451H452L453L454L455C456L457Y458M459E460Y461G462K463H464Q465N466L467Q468V469K470
(SEQ ID NO: 326)
(488) H423L424K425V426H427L428D429V430R431G432A433K434
(SEQ ID NO: 121)
(489) K425V426H427L428D429V430R431G432A433K434
(SEQ ID NO: 123)
(490) K425V426H427L428D429V430R431G432A433K434Q435Q436Q437Q438L439C440L441P442L443K444T445I446S447T448
S449F450T451H452
(SEQ ID NO: 327)
(491) K425V426H427L428D429V430R431G432A433K434Q435Q436Q437Q438L439C440L441P442L443K444T445I446S447T448
S449F450I451H452L453L454L455C456L457Y458M459E460Y461G462K463H464
(SEQ ID NO: 328)
(492) H427L428D429V430R431G432A433K434Q435Q436Q437Q438L439C440L441P442L443K444
(SEQ ID NO: 329)
(493) H427L428D429V430R431G432A433K434Q435Q436Q437Q438L439C440L441P442L443K444T445I446S447T448S449F450
T451H452L453L454L455C456L457Y458M459E460Y461G462K463H464Q465N466L467Q468V469K470
(SEQ ID NO: 330)
(494) K434Q435Q436Q437Q438L439C440L441P442L443K444T445I446S447T448S449F450T451H452
(SEQ ID NO: 331)
(495) K434Q435Q436Q437Q438L439C440L441P442L443K444T445I446S447T448S449F450T451H452L453L454L455C456L457
Y458M459E460Y461G462K463H464
(SEQ ID NO: 332)
(496) H452L453L454L455C456L457Y458M459E460Y461G462K463H464Q465N466L467Q468V469K470
(SEQ ID NO: 333)
(497) K463H464Q465N466L467Q468V469K470
(SEQ ID NO: 334)
Example 2 Determination of Very High Replikin Concentration at Accession No. NP 478030.
Replikin concentration was determined for a functionally undefined protein in the genome of an isolate of White Spot Syndrome Virus from 2000 made publicly available at NP 478030. The Replikin Concentration in the protein was again an unusually high 97.6 Replikin sequences per 100 amino acids. The Replikin concentration was determined by dividing the number of Replikin sequences identified in the amino acid sequence of the protein, 361 Replikin sequences, by the total amino acid length of the protein, 370 amino acids.
The amino acid sequence of NP 478030 is of interest because, like the protien in Example 1, it demonstrates an overlapping of Replikin sequences that results in very high Replikin concentration comparable to the highly-replicating P. falciparum of malaria. These high concentrations of Replikin sequences again provide a reservoir of Replikins for transfer to influenza viruses.
PubMed Accession Number NP 478030
Description: Protein of unknown function translated from complete genome sequence of the
shrimp white spot baculoform virus (WSSV) publicly available at PubMed Accession No.
NC003225.1.
Isolated Year: 2000
Source: Shrimp White Spot Syndrome Virus (Shrimp White Spot Baculoform Virus)
M1K2I3C4Q5I6S7S8P9T10L11T12L13S14I15P16L17E18G19V20Y21H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37
V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70
S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102
K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127
D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152
R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177
V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202
K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227
K228S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250L251K252
V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275K276Q277
N278P279W280R281K282N283L284C285L286L287K288K289N290V291K292S293A294K295Q296L297P298H299L300K301V302
L303L304D305V306R307G308A309K310Q311L312P313H314L315K316V317H318L319D320V321R322G323A324K325Q326Q327
Q328Q329L330C331L332P333L334K335T336I337S338T339S340F341T342H343L344L345L346C347L348Y349M350E351Y352
G353K354H355Q356N357L358Q359V360K361M362W363L364N365I366T367Y368T369S370
(SEQ ID NO: 335)
Replikin Sequences Identified in Accession No. ABS00973
Replikin Sequences Located in Amino-Terminal of Peptide
(1) H22V23K24Q25L26L27H28L29K30
(SEQ ID NO: 16)
(2) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36
(SEQ ID NO: 17)
(3) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39
(SEQ ID NO: 18)
(4) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45
(SEQ ID NO: 19)
(5) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53
K54
(SEQ ID NO: 20)
(6) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53
K54Q55N56P57W58R59K60
(SEQ ID NO: 21)
(7) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53
K54Q55N56P57W58R59K60N61L62C63L64L65K66
(SEQ ID NO: 22)
(8) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53
K54Q55N56P57W58R59K60N61L62C63L64L65K66K67
(SEQ ID NO: 23)
(9) H22V23K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53
K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70
(SEQ ID NO: 24)
(10) K24Q25L26L27H28L29K30
(SEQ ID NO: 10)
(11) K24Q25L26L27H28L29K30V31H32
(SEQ ID NO: 25)
(12) K24Q25L26L27H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43
(SEQ ID NO: 26)
(13) H28L29K30V31H32L33D34V35K36
(SEQ ID NO: 27)
(14) H28L29K30V31H32L33D34V35K36G37V38K39
(SEQ ID NO: 28)
(15) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45
(SEQ ID NO: 29)
(16) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54
(SEQ ID NO: 30)
(17) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57
W58R59K60
(SEQ ID NO: 31)
(18) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57
W58R59K60N61L62C63L64L65K66
(SEQ ID NO: 32)
(19) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57
W58R59K60N61L62C63L64L65K66K67
(SEQ ID NO: 33)
(20) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57
W58R59K60N61L62C63L64L65K66K67N68V69K70
(SEQ ID NO: 34)
(21) H28L29K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57
W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73
(SEQ ID NO: 35)
(22) K30V31H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59
K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 36)
(23) K30V31H32L33D34V35K36
(SEQ ID NO: 11)
(24) K30V31H32L33D34V35K36G37V38K39
(SEQ ID NO: 37)
(25) K30V31H32L33D34V35K36G37V38K39Q40L41L42H43
(SEQ ID NO: 12)
(26) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45
(SEQ ID NO: 38)
(27) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54
(SEQ ID NO: 39)
(28) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60
(SEQ ID NO: 40)
(29) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61
L62C63L64L65K66
(SEQ ID NO: 41)
(30) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61
L62C63L64L65K66K67
(SEQ ID NO: 42)
(31) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61
L62C63L64L65K66K67N68V69K70
(SEQ ID NO: 43)
(32) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61
L62C63L64L65K66K67N68V69K70S71A72K73
(SEQ ID NO: 44)
(33) H32L33D34V35K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61
L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79
(SEQ ID NO: 45)
(34) K36G37V38K39Q40L41L42H43L44K45
(SEQ ID NO: 46)
(35) K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65
K66K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 47)
(36) K36G37V38K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65
K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 48)
(37) K39Q40L41L42H43L44K45
(SEQ ID NO: 10)
(38) K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68
V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 49)
(39) K39Q40L41L42H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68
V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 50)
(40) H43L44K45V46R47L48D49V50R51G52A53K54
(SEQ ID NO: 51)
(41) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60
(SEQ ID NO: 52)
(42) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66
(SEQ ID NO: 53)
(43) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67
(SEQ ID NO: 54)
(44) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70
(SEQ ID NO: 55)
(45) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73
(SEQ ID NO: 56)
(46) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72
K73Q74L75P76H77L78K79
(SEQ ID NO: 57)
(47) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72
K73Q74L75P76H77L78K79V80H81L82D83V84K85
(SEQ ID NO: 58)
(48) H43L44K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72
K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88
(SEQ ID NO: 59)
(49) K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74
L75P76H77
(SEQ ID NO: 60)
(50) K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73
Q74L75P76H77L78K79V80H81
(SEQ ID NO: 61)
(51) K45V46R47L48D49V50R51G52A53K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74
L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 62)
(52) K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 63)
(53) K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 64)
(54) K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85
S86A87K88Q89L90P91H92
(SEQ ID NO: 65)
(55) K54Q55N56P57W58R59K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85
S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 66)
(56) K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91
H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 67)
(57) K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 68)
(58) K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 69)
(59) K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91
H92
(SEQ ID NO: 70)
(60) K60N61L62C63L64L65K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91
H92L93K94V95H96
(SEQ ID NO: 71)
(61) K66K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 13)
(62) K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 72)
(63) K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 73)
(64) K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 1)
(65) K66K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97
D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 74)
(66) K67N68V69K70S71A72K73Q74L75P76H77
(SEQ ID NO: 75)
(67) K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 76)
(68) K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 77)
(69) K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 78)
(70) K67N68V69K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98
V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 79)
(71) K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100
G101A102K103Q104L105P106H107
(SEQ ID NO: 80)
(72) K70S71A72K73Q74L75P76H77L78K79
(SEQ ID NO: 14)
(73) K70S71A72K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 81)
(74) K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 82)
(75) K70S71A72K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 83)
(76) K73Q74L75P76H77L78K79
(SEQ ID NO: 84)
(77) K73Q74L75P76H77L78K79V80H81
(SEQ ID NO: 85)
(78) K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102
K103Q104L105P106H107
(SEQ ID NO: 86)
(79) K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 87)
(80) K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 88)
(81) K73Q74L75P76H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102
K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122
(SEQ ID NO: 89)
(82) H77L78K79V80H81L82D83V84K85
(SEQ ID NO: 27)
(83) H77L78K79V80H81L82D83V84K85S86A87K88
(SEQ ID NO: 90)
(84) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94
(SEQ ID NO: 91)
(85) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103
(SEQ ID NO: 92)
(86) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105
P106H107L108K109
(SEQ ID NO: 93)
(87) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105
P106H107L108K109V110R111L112D113V114K115
(SEQ ID NO: 94)
(88) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105
P106H107L108K109V110R111L112D113V114K115S116A117K118
(SEQ ID NO: 95)
(89) H77L78K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105
P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124
(SEQ ID NO: 96)
(90) K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 97)
(91) K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122
(SEQ ID NO: 98)
(92) K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 99)
(93) K79V80H81L82D83V84K85
(SEQ ID NO: 11)
(94) K79V80H81L82D83V84K85S86A87K88
(SEQ ID NO: 100)
(95) K79V80H81L82D83V84K85S86A87K88Q89L90P91H92
(SEQ ID NO: 101)
(96) K79V80H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 102)
(97) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94
(SEQ ID NO: 103)
(98) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103
(SEQ ID NO: 104)
(99) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108
K109
(SEQ ID NO: 105)
(100) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108
K109V110R111L112D113V114K115
(SEQ ID NO: 106)
(101) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108
K109V110R111L112D113V114K115S116A117K118
(SEQ ID NO: 107)
(102) H81L82D83V84K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108
K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124
(SEQ ID NO: 108)
(103) K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 109)
(104) K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111
L112D113V114K115S116A117K118Q119L120P121H122
(SEQ ID NO: 110)
(105) K85S86A87K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111
L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 111)
(106) K85S86A87K88Q89L90P91H92L93K94
(SEQ ID NO: 14)
(107) K85S86A87K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 81)
(108) K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 112)
(109) K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114
K115S116A117K118Q119L120P121H122
(SEQ ID NO: 113)
(110) K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114
K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 114)
(111) K88Q89L90P91H92L93K94
(SEQ ID NO: 84)
(112) K88Q89L90P91H92L93K94V95H96
(SEQ ID NO: 85)
(113) K88Q89L90P91H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114
K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 115)
(114) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115
(SEQ ID NO: 116)
(115) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117
K118
(SEQ ID NO: 117)
(116) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117
K118Q119L120P121H122L123K124
(SEQ ID NO: 118)
(117) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117
K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133
(SEQ ID NO: 119)
(118) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117
K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139
(SEQ ID NO: 120)
(119) H92L93K94V95H96L97D98V99R100G101A102K103
(SEQ ID NO: 121)
(120) H92L93K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109
(SEQ ID NO: 122)
(121) K94V95H96L97D98V99R100G101A102K103
(SEQ ID NO: 123)
(122) K94V95H96L97D98V99R100G101A102K103Q104L105P106H107
(SEQ ID NO: 124)
(123) K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118
Q119L120P121H122
(SEQ ID NO: 125)
(124) K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118
Q119L120P121H122L123K124V125H126
(SEQ ID NO: 126)
(125) K94V95H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118
Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 127)
(126) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109
(SEQ ID NO: 128)
(127) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115
(SEQ ID NO: 129)
(128) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118
(SEQ ID NO: 130)
(129) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120
P121H122L123K124
(SEQ ID NO: 131)
(130) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120
P121H122L123K124V125H126L127D128V129R130G131A132K133
(SEQ ID NO: 132)
(131) H96L97D98V99R100G101A102K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120
P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139
(SEQ ID NO: 133)
(132) K103Q104L105P106H107L108K109
(SEQ ID NO: 84)
(133) K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122
(SEQ ID NO: 134)
(134) K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 135)
(135) K103Q104L105P106H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126
L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 136)
(136) H107L108K109V110R111L112D113V114K115
(SEQ ID NO: 137)
(137) H107L108K109V110R111L112D113V114K115S116A117K118
(SEQ ID NO: 138)
(138) H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124
(SEQ ID NO: 139)
(139) H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130
G131A132K133
(SEQ ID NO: 140)
(140) H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130
G131A132K133Q134L135P136H137L138K139
(SEQ ID NO: 141)
(141) H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130
G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148
(SEQ ID NO: 142)
(142) H107L108K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130
G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154
(SEQ ID NO: 143)
(143) K109V110R111L112D113V114K115S116A117K118Q119L120P121H122
(SEQ ID NO: 144)
(144) K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 145)
(145) K109V110R111L112D113V114K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132
K133Q134L135P136H137
(SEQ ID NO: 146)
(146) K115S116A117K118Q119L120P121H122L123K124
(SEQ ID NO: 14)
(147) K115S116A117K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 81)
(148) K115S116A117K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 109)
(149) K118Q119L120P121H122L123K124
(SEQ ID NO: 84)
(150) K118Q119L120P121H122L123K124V125H126
(SEQ ID NO: 85)
(151) K118Q119L120P121H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 112)
(152) H122L123K124V125H126L127D128V129R130G131A132K133
(SEQ ID NO: 121)
(153) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139
(SEQ ID NO: 122)
(154) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148
(SEQ ID NO: 147)
(155) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148Q149N150P151W152R153K154
(SEQ ID NO: 148)
(156) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
(SEQ ID NO: 149)
(157) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161
(SEQ ID NO: 150)
(158) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164
(SEQ ID NO: 151)
(159) H122L123K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145
G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167
(SEQ ID NO: 152)
(160) K124V125H126L127D128V129R130G131A132K133
(SEQ ID NO: 123)
(161) K124V125H126L127D128V129R130G131A132K133Q134L135P136H137
(SEQ ID NO: 124)
(162) K124V125H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147
K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 153)
Replikin Sequences Located in Mid-Molecule Portion of Peptide
(163) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139
(SEQ ID NO: 128)
(164) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148
(SEQ ID NO: 154)
(165) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154
(SEQ ID NO: 155)
(166) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154N155L156C157L158L159K160
(SEQ ID NO: 156)
(167) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154N155L156C157L158L159K160K161
(SEQ ID NO: 157)
(168) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164
(SEQ ID NO: 158)
(169) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167
(SEQ ID NO: 159)
(170) H126L127D128V129R130G131A132K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149
N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173
(SEQ ID NO: 160)
(171) K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156
C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 161)
(172) K133Q134L135P136H137L138K139
(SEQ ID NO: 84)
(173) K133Q134L135P136H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156
C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 162)
(174) H137L138K139V140R141L142D143V144R145G146A147K148
(SEQ ID NO: 51)
(175) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154
(SEQ ID NO: 52)
(176) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
(SEQ ID NO: 53)
(177) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161
(SEQ ID NO: 54)
(178) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161N162V163K164
(SEQ ID NO: 55)
(179) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161N162V163K164S165A166K167
(SEQ ID NO: 56)
(180) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161N162V163K164S165A166K167Q168L169P170H171L172K173
(SEQ ID NO: 57)
(181) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179
(SEQ ID NO: 58)
(182) H137L138K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160
K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182
(SEQ ID NO: 163)
(183) K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162
V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 60)
(184) K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162
V163K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 61)
(185) K139V140R141L142D143V144R145G146A147K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162
V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 164)
(186) K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 63)
(187) K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
L172K173V174H175
(SEQ ID NO: 64)
(188) K148Q149N150P151W152R153K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 165)
(189) K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177
V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201
(SEQ ID NO: 336)
(190) K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 68)
(191) K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 69)
(192) K154N155L156C157L158L159K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177
V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 167)
(193) K160K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 13)
(194) K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 72)
(195) K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183
L184L185H186
(SEQ ID NO: 2)
(196) K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183
L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201
(SEQ ID NO: 337)
(197) K160K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183
L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205
(SEQ ID NO: 338)
(198) K161N162V163K164S165A166K167Q168L169P170H171
(SEQ ID NO: 75)
(199) K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 76)
(200) K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184
L185H186
(SEQ ID NO: 170)
(201) K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184
L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201
(SEQ ID NO: 339)
(202) K161N162V163K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184
L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205
(SEQ ID NO: 340)
(203) K164S165A166K167Q168L169P170H171L172K173
(SEQ ID NO: 14)
(204) K164S165A166K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 81)
(205) K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 173)
(206) K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187
K188V189R190L191D192V193R194G195A196K197Q198L199P200H201
(SEQ ID NO: 341)
(207) K164S165A166K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187
K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205
(SEQ ID NO: 342)
(208) K167Q168L169P170H171L172K173
(SEQ ID NO: 84)
(209) K167Q168L169P170H171L172K173V174H175
(SEQ ID NO: 85)
(210) K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 176)
(211) K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190
L191D192V193R194G195A196K197Q198L199P200H201
(SEQ ID NO: 343)
(212) K167Q168L169P170H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190
L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205
(SEQ ID NO: 344)
(213) H171L172K173V174H175L176D177V178K179
(SEQ ID NO: 27)
(214) H171L172K173V174H175L176D177V178K179G180V181K182
(SEQ ID NO: 28)
(215) H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188
(SEQ ID NO: 29)
(216) H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194
G195A196K197Q198L199P200H201L202K203
(SEQ ID NO: 345)
(217) H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194
G195A196K197
(SEQ ID NO: 30)
(218) H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194
G195A196K197Q198L199P200H201L202K203V204H205L206D207V208R209G210A211K212
(SEQ ID NO: 346)
(219) H171L172K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194
G195A196K197Q198L199P200H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218
(SEQ ID NO: 347)
(220) K173V174H175L176D177V178K179
(SEQ ID NO: 11)
(221) K173V174H175L176D177V178K179G180V181K182
(SEQ ID NO: 37)
(222) K173V174H175L176D177V178K179G180V181K182Q183L184L185H186
(SEQ ID NO: 12)
(223) K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196
K197Q198L199P200H201
(SEQ ID NO: 348)
(224) K173V174H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196
K197Q198L199P200H201L202K203V204H205
(SEQ ID NO: 349)
(225) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198
L199P200H201L202K203V204H205L206D207V208R209G210A211K212
(SEQ ID NO: 350)
(226) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198
L199P200H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218
(SEQ ID NO: 351)
(227) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198
L199P200H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222
L223K224
(SEQ ID NO: 352)
(228) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198
L199P200H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222
L223K224K225
(SEQ ID NO: 353)
(229) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188
(SEQ ID NO: 38)
(230) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198
L199P200H201L202K203
(SEQ ID NO: 354)
(231) H175L176D177V178K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197
(SEQ ID NO: 39)
(232) K179G180V181K182Q183L184L185H186L187K188
(SEQ ID NO: 46)
(233) K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201
(SEQ ID NO: 355)
(234) K179G180V181K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202
K203V204H205
(SEQ ID NO: 356)
(235) K182Q183L184L185H186L187K188
(SEQ ID NO: 10)
(236) K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201
(SEQ ID NO: 357)
(237) K182Q183L184L185H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205
(SEQ ID NO: 358)
(238) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203
(SEQ ID NO: 359)
(239) H186L187K188V189R190L191D192V193R194G195A196K197
(SEQ ID NO: 51)
(240) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205L206D207V208R209
G210A211K212
(SEQ ID NO: 360)
(241) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205L206D207V208R209
G210A211K212Q213N214P215W216R217K218
(SEQ ID NO: 361)
(242) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205L206D207V208R209
G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224
(SEQ ID NO: 362)
(243) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205L206D207V208R209
G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225
(SEQ ID NO: 363)
(244) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205L206D207V208R209
G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227K228
(SEQ ID NO: 364)
(245) H186L187K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205L206D207V208R209
G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227K228S229A230K231
(SEQ ID NO: 365)
(246) K188V189R190L191D192V193R194G195A196K197Q198L199P200H201
(SEQ ID NO: 366)
(247) K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205
(SEQ ID NO: 317)
(248) K188V189R190L191D192V193R194G195A196K197Q198L199P200H201L202K203V204H205L206D207V208R209G210A211
K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227K228S229A230K231Q232L233P234H235
(SEQ ID NO: 275)
(249) K197Q198L199P200H201L202K203
(SEQ ID NO: 84)
(250) K197Q198L199P200H201L202K203V204H205
(SEQ ID NO: 85)
(251) K197Q198L199P200H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220
C221L222L223K224K225N226V227K228S229A230K231Q232L233P234H235
(SEQ ID NO: 224)
(252) H201L202K203V204H205L206D207V208R209G210A211K212
(SEQ ID NO: 121)
(253) H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218
(SEQ ID NO: 225)
(254) H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224
(SEQ ID NO: 226)
(255) H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224
K225
(SEQ ID NO: 227)
(256) H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224
K225N226V227K228
(SEQ ID NO: 228)
(257) H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224
K225N226V227K228S229A230K231
(SEQ ID NO: 229)
(258) H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224
K225N226V227K228S229A230K231Q232L233P234H235L236K237
(SEQ ID NO: 230)
(259) H201L202K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224
K225N226V227K228S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246
(SEQ ID NO: 231)
(260) K203V204H205L206D207V208R209G210A211K212
(SEQ ID NO: 123)
(261) K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226
V227K228S229A230K231Q232L233P234H235
(SEQ ID NO: 232)
(262) K203V204H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226
V227K228S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250
(SEQ ID NO: 233)
(263) H205L206D207V208R209G210A211K212Q213N214P215W216R217K218
(SEQ ID NO: 234)
(264) H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224
(SEQ ID NO: 235)
(265) H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225
(SEQ ID NO: 236)
(266) H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227K228
(SEQ ID NO: 237)
(267) H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227K228
S229A230K231
(SEQ ID NO: 238)
(268) H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227K228
S229A230K231Q232L233P234H235L236K237
(SEQ ID NO: 239)
(269) H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227K228
S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246
(SEQ ID NO: 240)
(270) H205L206D207V208R209G210A211K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227K228
S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250L251K252
(SEQ ID NO: 241)
(271) K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227K228S229A230K231Q232L233P234H235
(SEQ ID NO: 63)
(272) K212Q213N214P215W216R217K218N219L220C221L222L223K224K225N226V227K228S229A230K231Q232L233P234H235
L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250
(SEQ ID NO: 242)
(273) K218N219L220C221L222L223K224K225N226V227K228S229A230K231Q232L233P234H235L236K237V238L239L240D241
V242R243G244A245K246Q247L248P249H250
(SEQ ID NO: 243)
(274) K218N219L220C221L222L223K224K225N226V227K228S229A230K231Q232L233P234H235L236K237V238L239L240D241
V242R243G244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 244)
(275) K218N219L220C221L222L223K224K225N226V227K228S229A230K231Q232L233P234H235
(SEQ ID NO: 68)
(276) K224K225N226V227K228S229A230K231Q232L233P234H235
(SEQ ID NO: 13)
(277) K224K225N226V227K228S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247
L248P249H250
(SEQ ID NO: 275)
(278) K224K225N226V227K228S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247
L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 245)
(279) K225N226V227K228S229A230K231Q232L233P234H235
(SEQ ID NO: 75)
(280) K225N226V227K228S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247L248
P249H250
(SEQ ID NO: 247)
(281) K225N226V227K228S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247L248
P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 248)
(282) K228S229A230K231Q232L233P234H235L236K237
(SEQ ID NO: 14)
(283) K228S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250
(SEQ ID NO: 250)
(284) K228S229A230K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250L251
K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 251)
(285) K231Q232L233P234H235L236K237
(SEQ ID NO: 84)
(286) K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250
(SEQ ID NO: 253)
(287) K231Q232L233P234H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250L251K252V253L254
L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 254)
(288) H235L236K237V238L239L240D241V242R243G244A245K246
(SEQ ID NO: 256)
(289) H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250L251K252
(SEQ ID NO: 257)
(290) H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258
G259A260K261
(SEQ ID NO: 258)
(291) H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258
G259A260K261Q262L263P264H265L266K267
(SEQ ID NO: 259)
(292) H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258
G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275K276
(SEQ ID NO: 274)
(293) H235L236K237V238L239L240D241V242R243G244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258
G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282
(SEQ ID NO: 275)
(294) K237V238L239L240D241V242R243G244A245K246Q247L248P249H250
(SEQ ID NO: 262)
(295) K237V238L239L240D241V242R243G244A245K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260
K261Q262L263P264H265
(SEQ ID NO: 263)
(296) K246Q247L248P249H250L251K252
(SEQ ID NO: 84)
(297) K246Q247L248P249H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 253)
Replikin Sequences Located in Carboxy-Terminal Portion of Peptide
(298) H250L251K252V253L254L255D256V257R258G259A260K261
(SEQ ID NO: 256)
(299) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267
(SEQ ID NO: 257)
(300) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273
G274A275K276
(SEQ ID NO: 258)
(301) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273
G274A275K276Q277N278P279W280R281K282
(SEQ ID NO: 300)
(302) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273
G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288
(SEQ ID NO: 302)
(303) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273
G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288K289
(SEQ ID NO: 303)
(304) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273
G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288K289N290V291K292
(SEQ ID NO: 299)
(305) H250L251K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273
G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288K289N290V291K292S293A294K295
(SEQ ID NO: 301)
(306) K252V253L254L255D256V257R258G259A260K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275
K276Q277N278P279W280R281K282N283L284C285L286L287K288K289N290V291K292S293A294K295Q296L297P298H299
(SEQ ID NO: 304)
(307) K252V253L254L255D256V257R258G259A260K261Q262L263P264H265
(SEQ ID NO: 262)
(308) K261Q262L263P264H265L266K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282N283L284
C285L286L287K288K289N290V291K292S293A294K295Q296L297P298H299
(SEQ ID NO: 305)
(309) K261Q262L263P264H265L266K267
(SEQ ID NO: 84)
(310) H265L266K267V268L269L270D271V272R273G274A2755K276
(SEQ ID NO: 256)
(311) H265L266K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282
(SEQ ID NO: 307)
(312) H265L266K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288
(SEQ ID NO: 309)
(313) H265L266K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288
K289
(SEQ ID NO: 310)
(314) H265L266K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288
K289N290V291K292S293A294K295Q296L297P298H299L300K301
(SEQ ID NO: 311)
(315) H265L266K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288
K289N290V291K292
(SEQ ID NO: 306)
(316) H265L266K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288
K289N290V291K292S293A294K295
(SEQ ID NO: 308)
(317) H265L266K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288
K289N290V291K292S293A294K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310
(SEQ ID NO: 312)
(318) K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288K289N290
V291K292S293A294K295Q296L297P298H299
(SEQ ID NO: 313)
(319) K267V268L269L270D271V272R273G274A275K276Q277N278P279W280R281K282N283L284C285L286L287K288K289N290
V291K292S293A294K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314
(SEQ ID NO: 314)
(320) K276Q277N278P279W280R281K282N283L284C285L286L287K288K289N290V291K292S293A294K295Q296L297P298H299
(SEQ ID NO: 63)
(321) K276Q277N278P279W280R281K282N283L284C285L286L287K288K289N290V291K292S293A294K295Q296L297P298H299
L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314
(SEQ ID NO: 242)
(322) K276Q277N278P279W280R281K282N283L284C285L286L287K288K289N290V291K292S293A294K295Q296L297P298H299
L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314L315K316V317H318
(SEQ ID NO: 315)
(323) K282N283L284C285L286L287K288K289N290V291K292S293A294K295Q296L297P298H299
(SEQ ID NO: 68)
(324) K282N283L284C285L286L287K288K289N290V291K292S293A294K295Q296L297P298H299L300K301V302L303L304D305
V306R307G308A309K310Q311L312P313H314
(SEQ ID NO: 243)
(325) K282N283L284C285L286L287K288K289N290V291K292S293A294K295Q296L297P298H299L300K301V302L303L304D305
V306R307G308A309K310Q311L312P313H314L315K316V317H318
(SEQ ID NO: 316)
(326) K288K289N290V291K292S293A294K295Q296L297P298H299
(SEQ ID NO: 13)
(327) K288K289N290V291K292S293A294K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310Q311
L312P313H314
(SEQ ID NO: 3)
(328) K288K289N290V291K292S293A294K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310Q311
L312P313H314L315K316V317H318
(SEQ ID NO: 4)
(329) K289N290V291K292S293A294K295Q296L297P298H299
(SEQ ID NO: 75)
(330) K289N290V291K292S293A294K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310Q311L312
P313H314
(SEQ ID NO: 247)
(331) K289N290V291K292S293A294K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310Q311L312
P313H314L315K316V317H318
(SEQ ID NO: 318)
(332) K292S293A294K295Q296L297P298H299L300K301
(SEQ ID NO: 14)
(333) K292S293A294K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314
(SEQ ID NO: 250)
(334) K292S293A294K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314L315
K316V317H318
(SEQ ID NO: 319)
(335) K295Q296L297P298H299L300K301
(SEQ ID NO: 84)
(336) K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314
(SEQ ID NO: 253)
(337) K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314L315K316V317H318
(SEQ ID NO: 265)
(338) K295Q296L297P298H299L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314L315K316V317H318
L319D320V321R322G323A324K325Q326Q327Q328Q329L330C331L332P333L334K335T336I337S338T339S340F341T342H343
(SEQ ID NO: 320)
(339) H299L300K301V302L303L304D305V306R307G308A309K310
(SEQ ID NO: 256)
(340) H299L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314L315K316
(SEQ ID NO: 257)
(341) H299L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314L315K316V317H318L319D320V321R322
G323A324K325
(SEQ ID NO: 266)
(342) H299L300K301V302L303L304D305V306R307G308A309K310Q311L312P313H314L315K316V317H318L319D320V321R322
G323A324K325Q326Q327Q328Q329L330C331L332P333L334K335
(SEQ ID NO: 321)
(343) K301V302L303L304D305V306R307G308A309K310Q311L312P313H314
(SEQ ID NO: 262)
(344) K301V302L303L304D305V306R307G308A309K310Q311L312P313H314L315K316V317H318
(SEQ ID NO: 272)
(345) K301V302L303L304D305V306R307G308A309K310Q311L312P313H314L315K316V317H318L319D320V321R322G323A324
K325Q326Q327Q328Q329L330C331L332P333L334K335T336I337S338T339S340F341T342H343
(SEQ ID NO: 322)
(346) K310Q311L312P313H314L315K316
(SEQ ID NO: 84)
(347) K310Q311L312P313H314L315K316V317H318
(SEQ ID NO: 85)
(348) K310Q311L312P313H314L315K316V317H318L319D320V321R322G323A324K325Q326Q327Q328Q329L330C331L332P333
L334K335T336I337S338T339S340F341T342H343
(SEQ ID NO: 323)
(349) K310Q311L312P313H314L315K316V317H318L319D320V321R322G323A324K325Q326Q327Q328Q329L330C331L332P333
L334K335T336I337S338T339S340F341T342H343L344L345L346C347L348Y349M350E351Y352G353K354H355
(SEQ ID NO: 324)
(350) H314L315K316V317H318L319D320V321R322G323A324K325
(SEQ ID NO: 121)
(351) H314L315K316V317H318L319D320V321R322G323A324K325Q326Q327Q328Q329L330C331L332P333L334K335
(SEQ ID NO: 325)
(352) H314L315K316V317H318L319D320V321R322G323A324K325Q326Q327Q328Q329L330C331L332P333L334K335T336I337
S338T339S340F341T342H343L344L345L346C347L348Y349M350E351Y352G353K354H355Q356N357L358Q359V360K361
(SEQ ID NO: 326)
(353) K316V317H318L319D320V321R322G323A324K325Q326Q327Q328Q329L330C331L332P333L334K335T336I337S338T339
S340F341T342H343L344L345L346C347L348Y349M350E351Y352G353K354H355
(SEQ ID NO: 328)
(354) K316V317H318L319D320V321R322G323A324K325
(SEQ ID NO: 123)
(355) K316V317H318L319D320V321R322G323A324K325Q326Q327Q328Q329L330C331L332P333L334K335T336I337S338T339
S340F341T342H343
(SEQ ID NO: 327)
(356) H318L319D320V321R322G323A324K325Q326Q327Q328Q329L330C331L332P333L334K335
(SEQ ID NO: 329)
(357) H318L319D320V321R322G323A324K325Q326Q327Q328Q329L330C331L332P333L334K335T336I337S338T339S340F341
T342H343L344L345L346C347L348Y349M350E351Y352G353K354H355Q356N357L358Q359V360K361
(SEQ ID NO: 330)
(358) K325Q326Q327Q328Q329L330C331L332P333L334K335T336I337S338T339S340F341T342H343
(SEQ ID NO: 331)
(359) K325Q326Q327Q328Q329L330C331L332P333L334K335T336I337S338T339S340F341T342H343L344L345L346C347L348
Y349M350E351Y352G353K354H355
(SEQ ID NO: 332)
(360) H343L344L345L346C347L348Y349M350E351Y352G353K354H355Q356N357L358Q359V360K361
(SEQ ID NO: 333)
(361) K354H355Q356N357L358Q359V360K361
(SEQ ID NO: 334)
Example 3 Determination of Low Replikin Concentrations in 2005 and 2007 Isolates of WSSV from Accession Nos. AAW88445 and ABS00973
In 2006 and 2007 White Spot Syndrome Virus (WSSV) has been observed to be dormant in shrimp. This continued decline of WSSV into “quiescent” or “dormant” levels in 2006-2007 is demonstrated in mean Replikin concentrations for viruses isolated during 2005-2007 that are very low as compared to years wherein the virus demonstrated greater virulence, such as 2001. See Table 4 in Example 4 below. The continued quiescence in WSSV in 2007 may be contrasted with an observed rising of Replikin concentration in Taura Syndrome Virus Replikin during this period.
As may be seen from the analysis below, Accession Nos. AAW88445 and ABS00973 have low observed Replikin concentrations. ABS00973 contains a single Replikin sequence in the entire disclosed amino acid sequence of 240 residues. The single Replikin sequence is underlined. The Replikin concentration of Accession No. ABS00973 is an inordinately low 0.5.
Replikin Analysis
PubMed Code: AAW88445
Description:
Isolated Year: 2005
Source: Shrimp White Spot Syndrome Virus
M1S2N3G4A5T6I7S8D9E10R11L12I13L14I15L16D17K18I19V20E21R22R23G24V25S26N27L28S29E30L31L32I33H34P35I36T37
K38H39I40N41E42L43L44K45N46T47V48R49H50G51D52R53V54Y55M56K57D58A59E60L61D62V63R64S65R66L67E68D69I70K71
K72D73C74V75L76K77A78I79E80K81Q82G83I84D85V86R87Q88I89I90T91D92Y93L94A95K96R97K98L99T100Q101N102L103
V104H105W106Y107R108P109P110I111S112C113T114D115I116D117E118K119I120Q121Q122E123T124G125Q126V127G128
R129C130S131V132A133T134Y135N136L137R138I139G140G141D142D143G144E145F146T147R148Y149D150F151S152I153
P154L155G156D157F158K159I160T161A162K163L164F165R166S167I168N169D170E171D172V173D174A175V176I177L178
V179S180R181S182D183V184V185N186D187V188L189S190F191E192A193F194N195R196T197G198E199R200V201V202I203
F204F205N206V207I208V209E210G211K212S213K214D215I216D217I218V219C220K221S222R223Y224K225H226T227H228
I229L230N231G232E233S234A235T236Y237A238V239K240R241I242K243R244G245D246T247R248D249D250I251L252F253
A254I255T256A257F258K259E260E261
(SEQ ID NO: 367)
Replikin Sequences in Amino-terminal Portion of Peptide
(1) H34P35I36T37K38H39I40N41E42L43L44K45N46T47V48R49H50G51D52R53V54Y55M56K57D58A59E60L61D62V63R64S65
R66L67E68D69I70K71K72D73C74V75L76K77
(SEQ ID NO: 431)
(2) H34P35I36T37K38H39I40N41E42L43L44K45
(SEQ ID NO: 432)
(3) H34P35I36T37K38H39I40N41E42L43L44K45N46T47V48R49H50G51D52R53V54Y55M56K57D58A59E60L61D62V63R64S65
R66L67E68D69I70K71K72D73C74V75L76K77A78I79E80K81
(SEQ ID NO: 433)
(4) K38H39I40N41E42L43L44K45
(SEQ ID NO: 434)
(5) K38H39I40N41E42L43L44K45N46T47V48R49H50
(SEQ ID NO: 435)
(6) H39I40N41E42L43L44K45N46T47V48R49H50G51D52R53V54Y55M56K57D58A59E60L61D62V63R64S65R66L67E68D69I70
K71K72D73C74V75L76K77
(SEQ ID NO: 436)
(7) H39I40N41E42L43L44K45N46T47V48R49H50G51D52R53V54Y55M56K57D58A59E60L61D62V63R64S65R66L67E68D69I70
K71K72D73C74V75L76K77A78I79E80K81
(SEQ ID NO: 437)
(8) H50G51D52R53V54Y55M56K57D58A59E60L61D62V63R64S65R66L67E68D69I70K71K72D73C74V75L76K77
(SEQ ID NO: 438)
(9) H50G51D52R53V54Y55M56K57D58A59E60L61D62V63R64S65R66L67E68D69I70K71K72D73C74V75L76K77A78I79E80K81
(SEQ ID NO: 439)
(10) K71K72D73C74V75L76K77A78I79E80K81Q82G83I84D85V86R87Q88I89I90T91D92Y93L94A95K96R97K98L99T100Q101
N102L103V104H105
(SEQ ID NO: 440)
(11) K72D73C74V75L76K77A78I79E80K81Q82G83I84D85V86R87Q88I89I90T91D92Y93L94A95K96R97K98L99T100Q101N102
L103V104H105
(SEQ ID NO: 441)
Replikin Sequences in Mid-Molecule Portion of Peptide
Zero Replikins.
Replikin Sequences in Carboxy-Terminal Portion of Peptide
(12) K212S213K214D215I216D217I218V219C220K221S222R223Y224K225H226
(SEQ ID NO: 442)
(13) K212S213K214D215I216D217I218V219C220K221S222R223Y224K225H226I227H228
(SEQ ID NO: 443)
(14) K214D215I216D217I218V219C220K221S222R223Y224K225H226
(SEQ ID NO: 444)
(15) K214D215I216D217I218V219C220K221S222R223Y224K225H226I227H228
(SEQ ID NO: 445)
Replikin Count = Number of Replikins per 100 amino acids = 15/261 = 5.7
PubMed Code: ABS00973
Description:
Isolated: 2007
Source: Shrimp white spot syndrome virus
M1D2L3S4F5T6L7S8V9V10S11A12I13L14A15I16T17A18V19I20A21V22F23I24V25I26F27R28Y29H30N31T32V33T34K35T36I37
E38T39 H40T41G42N43I44E45T46N47M48D49E50N51L52R53I54P55V56T57A58E59V60G61S62G63Y64F65K66M67T68D69V70S71
F72D73S74D75T76L77G78K79I80K81I82R83N84G85K86 S87D88A89Q90M91K92E93E94D95A96D97L98V99I100T101P102V103
E104G105R106A107L108E109V110T111V112G113Q114N115L116T117F118E119G120T121F122K123M124W125N126N127T128
S129R130K131I132N133I134T135G136M137Q138M139V140P141K142I143N144P145S146K147A148F149V150G151S152S153
N154T155S156S157F158T159P160V161S162I163D164E165D166E167V168G169T170F171V172C173G174T175T176F177G178
A179P180I181A182A183T184A185G186G187N188L189F190D191M192Y193V194H195V196T197Y198S199G200T201E202T203
E204
(SEQ ID NO: 446)
Replikin Sequences in Amino-Terminal Portion of Peptide
(1) H40T41G42N43I44E45T46N47M48D49E50N51L52R53I54P55V56T57A58E59V60G61S62G63Y64F65K66M67T68D69V70
S71F72D73S74D75T76L77G78K79I80K81I82R83N84G85K86
(SEQ ID NO: 447)
Mid-molecule: Zero Replikins.
Carboxy-terminal: Zero Replikins.
Replikin Count = Number of Replikins per 100 amino acids = 1/204 = 0.5
Example 4 Determination of Replikin Concentration in Publicly Available Accession Numbers for Isolates of WSSV from 1995 through 2007
Mean Replikin concentrations were determined for all amino acid sequences for WSSV with accession numbers publicly available at www.pubmed.com. The amino acid sequences were scanned for Replikin sequences of 7 to 50 amino acids comprising (1) at least one lysine residue located at a first terminus of the sequence and at least one lysine residue or at least one histidine residue located at a second terminus of the sequence; (2) a first lysine residue located six to ten residues from a second lysine residue; (3) at least one histidine residue; and (4) at least 6% lysine residues. The total number of Replikin sequences was determined for each available accession number. The total number of Replikin sequences in each accession number was then divided by the total number of amino acid residues disclosed in the accession number. The result was the Replikin concentration. The mean Replikin concentration was then determined for all viruses isolated and reported in a particular year. Table 4 provides the results of the Replikin concentration analysis.
TABLE 4
WSSV Replikin Concentration by Year
Year PubMed Accession Number-Replikin Count
1995 CAA88950 18 CAA91970 59
No. of isolates: 2 Mean Replikin Count: 4.4 Standard Deviation: 0.6 Significance: low p < 0.10
1996 CAE17687 160 CAB03144 29 CAB03173 31
No. of isolates: 3 Mean Replikin Count: 6.0 Standard Deviation: 2.6 Significance: low p > 0.50,
prev p < 0.30
1997
1998 ABA54417 48
No. of isolates: 1 Mean Replikin Count: 6.2 Standard Deviation: 0.0 Significance: prev p > 0.50
1999
2000 NP_478030 361 NP_478019 361 NP_478001 361 NP_477774 361 NP_477756 361 NP_477753
361 NP_477809 361 NP_477768 361 NP_477523 361 NP_477959 361 NP_478053 361
NP_478052 361 NP_478051 361 NP_478050 361 NP_478049 361 NP_478048 361 NP_478047
361 NP_478046 361 NP_478045 361 NP_478044 361 NP_478043 361 NP_478042 361
NP_478041 361 NP_478039 361 NP_478038 361 NP_478037 361 NP_478036 361 NP_478035
361 NP_478034 361 NP_478033 361 NP_478032 361 NP_478031 361 NP_478029 361
NP_478028 361 NP_478027 361 NP_478026 361 NP_478025 361 NP_478024 361 NP_478023
361 NP_478022 361 NP_478021 361 NP_478020 361 NP_478018 361 NP_478017 361
NP_478016 361 NP_478015 361 NP_478014 361 NP_478013 361 NP_478012 361 NP_478011
361 NP_478010 361 NP_478009 361 NP_478008 361 NP_478007 361 NP_478006 361
NP_478005 361 NP_478004 361 NP_478003 361 NP_478002 361 NP_478000 361 NP_477999
361 NP_477998 361 NP_477997 361 NP_477996 361 NP_477995 361 NP_477994 361
NP_477993 361 NP_477992 361 NP_477991 361 NP_477990 361 NP_477989 361 NP_477988
361 NP_477987 361 NP_477986 361 NP_477985 361 NP_477984 361 NP_477983 361
NP_477982 361 NP_477981 361 NP_477980 361 NP_477979 361 NP_477978 361 NP_477977
361 NP_477976 361 NP_477975 361 NP_477974 361 NP_477973 361 NP_477972 361
NP_477971 361 NP_477970 361 NP_477969 361 NP_477968 361 NP_477967 361 NP_477966
361 NP_477965 361 NP_477964 361 NP_477963 361 NP_477962 361 NP_477961 361
NP_477960 361 NP_477958 361 NP_477957 361 NP_477956 361 NP_477955 361 NP_477954
361 NP_477953 361 NP_477952 361 NP_477951 361 NP_477950 361 NP_477949 361
NP_477948 361 NP_477947 361 NP_477946 361 NP_477945 361 NP_477944 361 NP_477943
361 NP_477942 361 NP_477941 361 NP_477940 361 NP_477939 361 NP_477938 361
NP_477937 361 NP_477936 361 NP_477935 361 NP_477934 361 NP_477933 361 NP_477932
361 NP_477931 361 NP_477930 361 NP_477929 361 NP_477928 361 NP_477927 361
NP_477926 361 NP_477925 361 NP_477924 361 NP_477923 361 NP_477922 361 NP_477921
361 NP_477920 361 NP_477919 361 NP_477918 361 NP_477917 361 NP_477916 361
NP_477915 361 NP_477914 361 NP_477913 361 NP_477912 361 NP_477911 361 NP_477910
361 NP_477909 361 NP_477908 361 NP_477907 361 NP_477906 361 NP_477905 361
NP_477904 361 NP_477903 361 NP_477902 361 NP_477901 361 NP_477900 361 NP_477899
361 NP_477898 361 NP_477897 361 NP_477896 361 NP_477895 361 NP_477894 361
NP_477893 361 NP_477892 361 NP_477891 361 NP_477890 361 NP_477889 361 NP_477888
361 NP_477887 361 NP_477886 361 NP_477885 361 NP_477884 361 NP_477883 361
NP_477882 361 NP_477881 361 NP_477880 361 NP_477879 361 NP_477878 361 NP_477877
361 NP_477876 361 NP_477875 361 NP_477874 361 NP_477873 361 NP_477872 361
NP_477871 361 NP_477870 361 NP_477869 361 NP_477868 361 NP_477867 361 NP_477866
361 NP_477865 361 NP_477864 361 NP_477863 361 NP_477862 361 NP_477861 361
NP_477860 361 NP_477859 361 NP_477858 361 NP_477857 361 NP_477856 361 NP_477855
361 NP_477854 361 NP_477853 361 NP_477852 361 NP_477851 361 NP_477850 361
NP_477849 361 NP_477848 361 NP_477847 361 NP_477846 361 NP_477845 361 NP_477844
361 NP_477843 361 NP_477842 361 NP_477841 361 NP_477840 361 NP_477839 361
NP_477838 361 NP_477837 361 NP_477836 361 NP_477835 361 NP_477834 361 NP_477833
361 NP_477832 361 NP_477831 361 NP_477830 361 NP_477829 361 NP_477828 361
NP_477827 361 NP_477826 361 NP_477825 361 NP_477824 361 NP_477823 361 NP_477822
361 NP_477821 361 NP_477820 361 NP_477819 361 NP_477818 361 NP_477817 361
NP_477816 361 NP_477815 361 NP_477814 361 NP_477813 361 NP_477812 361 NP_477811
361 NP_477810 361 NP_477808 361 NP_477807 361 NP_477806 361 NP_477805 361
NP_477804 361 NP_477803 361 NP_477802 361 NP_477801 361 NP_477800 361 NP_477799
361 NP_477798 361 NP_477797 361 NP_477796 361 NP_477795 361 NP_477794 361
NP_477793 361 NP_477792 361 NP_477791 361 NP_477790 361 NP_477789 361 NP_477788
361 NP_477787 361 NP_477786 361 NP_477785 361 NP_477784 361 NP_477783 361
NP_477782 361 NP_477781 361 NP_477780 361 NP_477779 361 NP_477778 361 NP_477777
361 NP_477776 361 NP_477775 361 NP_477773 361 NP_477772 361 NP_477771 361
NP_477770 361 NP_477769 361 NP_477767 361 NP_477766 361 NP_477765 361 NP_477764
361 NP_477763 361 NP_477762 361 NP_477761 361 NP_477760 361 NP_477759 361
NP_477758 361 NP_477757 361 NP_477755 361 NP_477754 361 NP_477752 361 NP_477751
361 NP_477750 361 NP_477749 361 NP_477748 361 NP_477747 361 NP_477746 361
NP_477745 361 NP_477744 361 NP_477743 361 NP_477742 361 NP_477741 361 NP_477740
361 NP_477739 361 NP_477738 361 NP_477737 361 NP_477736 361 NP_477735 361
NP_477734 361 NP_477733 361 NP_477732 361 NP_477731 361 NP_477730 361 NP_477729
361 NP_477728 361 NP_477727 361 NP_477726 361 NP_477725 361 NP_477724 361
NP_477723 361 NP_477722 361 NP_477721 361 NP_477720 361 NP_477719 361 NP_477718
361 NP_477717 361 NP_477716 361 NP_477715 361 NP_477714 361 NP_477713 361
NP_477712 361 NP_477711 361 NP_477710 361 NP_477709 361 NP_477708 361 NP_477707
361 NP_477706 361 NP_477705 361 NP_477704 361 NP_477703 361 NP_477702 361
NP_477701 361 NP_477700 361 NP_477699 361 NP_477698 361 NP_477697 361 NP_477696
361 NP_477695 361 NP_477694 361 NP_477693 361 NP_477692 361 NP_477691 361
NP_477690 361 NP_477689 361 NP_477688 361 NP_477687 361 NP_477686 361 NP_477685
361 NP_477684 361 NP_477683 361 NP_477682 361 NP_477681 361 NP_477680 361
NP_477679 361 NP_477678 361 NP_477677 361 NP_477676 361 NP_477675 361 NP_477674
361 NP_477673 361 NP_477672 361 NP_477671 361 NP_477670 361 NP_477669 361
NP_477668 361 NP_477667 361 NP_477666 361 NP_477665 361 NP_477664 361 NP_477663
361 NP_477662 361 NP_477661 361 NP_477660 361 NP_477659 361 NP_477658 361
NP_477657 361 NP_477656 361 NP_477655 361 NP_477654 361 NP_477653 361 NP_477652
361 NP_477651 361 NP_477650 361 NP_477649 361 NP_477648 361 NP_477647 361
NP_477646 361 NP_477645 361 NP_477644 361 NP_477643 361 NP_477642 361 NP_477641
361 NP_477640 361 NP_477639 361 NP_477638 361 NP_477637 361 NP_477636 361
NP_477635 361 NP_477634 361 NP_477633 361 NP_477632 361 NP_477631 361 NP_477630
361 NP_477629 361 NP_477628 361 NP_477627 361 NP_477626 361 NP_477625 361
NP_477624 361 NP_477623 361 NP_477622 361 NP_477621 361 NP_477620 361 NP_477619
361 NP_477618 361 NP_477617 361 NP_477616 361 NP_477615 361 NP_477614 361
NP_477613 361 NP_477612 361 NP_477611 361 NP_477610 361 NP_477609 361 NP_477608
361 NP_477607 361 NP_477606 361 NP_477605 361 NP_477604 361 NP_477603 361
NP_477602 361 NP_477601 361 NP_477600 361 NP_477599 361 NP_477598 361 NP_477597
361 NP_477596 361 NP_477595 361 NP_477594 361 NP_477593 361 NP_477592 361
NP_477591 361 NP_477590 361 NP_477589 361 NP_477588 361 NP_477587 361 NP_477586
361 NP_477585 361 NP_477584 361 NP_477583 361 NP_477582 361 NP_477581 361
NP_477580 361 NP_477579 361 NP_477578 361 NP_477577 361 NP_477576 361 NP_477575
361 NP_477574 361 NP_477573 361 NP_477572 361 NP_477571 361 NP_477570 361
NP_477569 361 NP_477568 361 NP_477567 361 NP_477566 361 NP_477565 361 NP_477564
361 NP_477563 361 NP_477562 361 NP_477561 361 NP_477560 361 NP_477559 361
NP_477558 361 NP_477557 361 NP_477556 361 NP_477555 361 NP_477554 361 NP_477553
361 NP_477552 361 NP_477551 361 NP_477550 361 NP_477549 361 NP_477548 361
NP_477547 361 NP_477546 361 NP_477545 361 NP_477544 361 NP_477543 361 NP_477542
361 NP_477541 361 NP_477540 361 NP_477539 361 NP_477538 361 NP_477537 361
NP_477536 361 NP_477535 361 NP_477534 361 NP_477533 361 NP_477532 361 NP_477531
361 NP_477530 361 NP_477529 361 NP_477527 361 NP_477526 361 NP_477525 361
NP_477524 361
No. of isolates: 529 Mean Replikin Count: 97.6 Std. Deviation: 0.0 Significance: low p < 0.001
2002
2003
2004
2005 AAZ29239 9 XP_001681561 6
No. of isolates: 2 Mean Replikin Count: 2.6 Std. Deviation: 2.4 Significance: low p > 0.20,
prev p < 0.001
2006 ABM92267 14 ABP01348 1 ABM64218 6 ABI34434 6 ABI93178 4 ABI93177 3 ABI93176 6
ABI93174 12 ABQ12866 3 ABD65308 2 ABD65303 1 ABD65302 4 ABD65300 3 ABD65298 1
No. of isolates: 14 Mean Replikin Count: 2.7 Std. Deviation: 2.5 Significance: low p < 0.001,
prev p > 0.50
2007 2ED6_L 1 2ED6_K 1 2ED6_J 1 2ED6_I 1 2ED6_H 1 2ED6_G 1 2ED6_F 1 2ED6_E 1 2ED6_D 1
2ED6_C 1 2ED6_B 1 2ED6_A 1 ABQ12772 15 ABQ12773 3 ABQ12771 6 ABQ12770 9
ABO69369 2 ABO69368 2 ABS00974 5 ABS00973 1 ABQ44211 3 ABQ44210 4 ABP52058 4
ABP52057 1 ABP52054 5
No. of isolates: 25 Mean Replikin Count: 1.3 Std. Deviation: 1.2 Significance: low p < 0.001,
prev p < 0.05
Example 5 Determination of Replikin Concentrations in 2001 and 2005 Isolates of TSV Publicly Available at Accession Nos. AAM73766 and AAY89096
The Taura Syndrome Shrimp Virus is less virulent than WSSV and the structure of the TSV Replikin Scaffold is less closely related to influenza virus than are the structures of WSSV Replikin Scaffolds. In year 2000, TSV had a Replikin concentration of 3.3. Between 2001 and 2004, TSV had a lower mean Replikin concentration, as low as 0.7, and its Replikin Scaffold disappeared. In 2005 the Replikin Scaffold reappeared, with an increase in lysines and histidines, and a commensurate increase in Replikin concentration to 3.9, followed by an increase in TSV outbreaks in 2006-2007.
Below is a comparison of the Replikin Scaffold identified in AAK72220 in an isolate of TSV from 2000 and the Replikin Scaffold identified in AAY89096 in an isolate of TSV from 2005. The TSV Replikin Scaffolds are also compared to two Replikin Scaffolds in H1N1 influenza virus in the 1918 pandemic and shrimp WSSV in 2000.
Figure US08050871-20111101-C00012
 		(SEQ ID NO: 384) (SEQ ID NO: 448) (SEQ ID NO: 7) (SEQ ID NO: 6) 1918 H1N1 Human Influenza Pandemic 1918 H1N1 Human Influenza Pandemic 2000 Shrimp White Spot Syndrome Virus 2000 Shrimp White Spot Syndrome Virus
kkvqanktrvfaasnqglalalrryylsfldh (SEQ ID NO: 8) 2000 Taura Syndrome Virus AAK72220
kkacrnagykeaclheldcksfllaqqgragah (SEQ ID NO: 9) 2005 Taura Syndrome Virus AAY89096
The following analysis of Accession Nos. AAM73766 and AAY89096 demonstrate Replikin concentration analysis of amino acid sequences of isolates of Taura Syndrome Virus having publicly available accession numbers at www.pubmed.com.
PubMed Code: AAM73766
Description:
Isolated: 2001
Source: Taura syndrome virus
M1P2A3N4P5V6E7I8D9N10F11D12T13T14T15S16G17G18L19I20P21G22G23S24V25T26N27S28E29G30S31T32I33L34M35N36D37
I38P39I40T41N42Q43N44V45V46L47S48K49N50V51T52D53N54L55F56E57V58Q59D60Q61A62L63I64E65S66L67S68R69D70V71
L72L73H74N75D76S77W78T79S80S81D82D83E84I85G86T87T88M89T90Q91E92Q93L94A95T96E97F98N99Q100P101H102L103
Y104E105I106S107L108P109D110D111I112V113R114K115S116L117F118M119S120N121K122L123A124N125I126A127Y128
M129R130C131D132Y133E134V135T136V137R138V139Q140A141T142P143F144L145Q146G147A148L149W150L151W152N153
K154M155N156A157K158Q159T160S161I162I163R164R165T166L167T168E169H170L171R172S173I174T175S176F177P178
G179I180E181M182N183L184Q185S186E187A188R189A190I191T192L193S194I195P196Y197T198S199E200F201Q202V203
F204N205P206R207N208V209N210N211L212N213S214I215R216L217S218V219L220S221Q222L223Q224G225P226E227D228
V229E230S231A232S233Y234S235I236Y237G238R239L240K241N242I243K244L245Y246G247H248A249P250S251V252T253
S254S255V256Y257P258S259T260Q261S262G263Y264D265D266D267C268P269I270V271H272A273G274T275D276E277D278
S279S280K281Q282G283I284V285S286R287V288A289D290T291V292G293A294V295A296N297V298V299D300G301V302G303
V304P305I306L307S308T309I310A311K312P313V314S315W316V317S318G319V320V321S322N323V324A325S326M327F328
G329F330S331K332D333R334D335M336T337K338V339N340A341Y342E343N344L345P346G347K348G349F350T351H352G353
V354G355F356D357Y358G359V360P361L362S363L364F365P366N367N368A369I370D371P372T373I374A375V376P377E378
G379L389D381E382M383S384I385E386Y387L388A389Q390R391P392Y393M394L395N396R397Y398T399I400R401G402G403
D404T405P406D407V408H409G410T411I412V413A414D415I416P417V418S419P420V421N422F423S424L425Y426G427K428
V429I430A431K432Y433R434T435L436F437A438A439P440V441S442L443A444V445A446M447A448N449W450W451R452G453
N454I455N456L457N458L459R460F461A462K463T464Q465Y466H467Q468C469R470L471L472V473Q474Y475L476P477Y478
G479S480G481V482Q483P484I485E486S487I488L489S490Q491I492I493D494I495S496Q497V498D499D500K501G502I503
D504I505A506F507P508S509V510Y511P512N513K514W515M516R517V518Y519D520P521A522K523V524G525Y526T527A528
D529C530A531P532G533R534I535V536I537S538V539L540N541P542L543I544S545A546S547T548V549S550P551N552I553
V554M555Y556P557W558V559H560W561S562N563L564E565V566A567E568P569G570T571L572A573K574A575A576I577G578
F579N580Y581P582A583D584V585P586E587E588P589T590F591S592V593T594R595A596P597V598S599G600T601L602F603
T604L605L606Q607D608T609K610V611S612L613G614E615A616D617G618V619F620S621L622Y623F624T625N626T627T628
T629G630R631R632H633R634L635T636Y637A638G639L640P641G642E643L644G645S646C647E648I649V650K651L652P653
Q654G655Q656Y657S658I659E660Y661A662A663T664S665A666P667T668L669V670L671D672R673P674I675F676S677E678
P679I680G681P682K683Y684V685V686T687K688V689K690N691G692D693V694V695S696I697S698E699E700T701L702V703
T704C705G706S707M708A709A710L711G712G713A714T715V716A717L718Q719S720V721D722E723T724I725E726I727L728
K729L730E731S732D733F734E735S736K737A738P739V740K741F742T743P744G745N746Y747T748V749V750T751E752A753
S754D755V756E757L758V759T760N761Q762D763I764T765V766N767E768H769N770P771R772T773H774A775G776I777D778
E779E780P781P782V783K784R785S786V787I788G789R790I791V792R793R794V795A796R797Y798V799P800N801K802L803
I804R805R806I807L808R809D810L811S812Q813S814P815C816I817Y818P819S820T821H822A823G824L825D826Y827S828
S829S830D831T832S833T834M835L836T837T838M839G840E841Q842F843V844S845L846R847M848L849T850R851R852S853
S854P855V856D857I858L859R860G861D862L863V864T865L866P867G868I869S870F871G872T873D874N875S876L877R878
Q879S880L881V882N883I884I885S886Y887M888Y889R890F891T892H893G894S895I896S897Y898K899I900I901P902K903
N904K905G906D907L908Y909I910T911T912T913S914P915D916S917I918E919T920S921T922S923A924Y925Q926F927D928
T929N930R931A932M933H934Y935I936N937T938S939L940N941P942M943A944Q945I946S947L948P949Y950Y951S952P953
A954E955N956L957V958I959D960S961K962S963F964P965Q966L967S968D969L970S971I972S973N974L975E976R977T978
E979N980E981Y982F983V984L985A986S987A988G989D990D991H992T993F994S995Q996L997A998G999C1000P1001A1002
F1003T1004F1005G1006P1007A1008E1009L1010A1011
(SEQ ID NO: 449)
Replikin Sequences in Amino-Terminal Portion of Peptide
(1) H102L103Y104E105I106S107L108P109D110D111I112V113R114K115S116L117F118M119S120N121K122
(SEQ ID NO: 450)
(2) K332D333R334D335M336T337K338V339N340A341Y342E343N344L345P346G347K348G349F350T351H352
(SEQ ID NO: 451)
(3) K338V339N340A341Y342E343N344L345P346G347K348G349F350T351H352
(SEQ ID NO: 452)
Replikin Sequences in Mid-Molecule Portion of Peptide
Zero Replikins.
Replikin Sequences in Carboxy-Terminal Portion of Peptide
(4) K729L730E731S732D733F734E735S736K737A738P739V740K741F742T743P744G745N746Y747T748V749V750T751E752
A753S754D755V756E757L758V759T760N761Q762D763I764T765V766N767E768H769N770P771R772T773H774
(SEQ ID NO: 453)
(5) K729L730L731S732D733F734L735S736K737A738P739V740K741F742T743P744G745N746Y747T748V749V750T751E752
A753S754D755V756E757L758V759T760N761Q762D763I764T765V766N767E768H769
(SEQ ID NO: 454)
(6) H893G894S895I896S897Y898K899I900I901P902K903N904K905
(SEQ ID NO: 455)
(7) K899I900I901P902K903N904K905G906D907L908Y909I910T911T912T913S914P915D916S917I918E919T920S921T922
S923A924Y925Q926F927D928T929N930R931A932M933H934
(SEQ ID NO: 456)
Replikin Count = Number of Replikins per 100 amino acids = 7/1011 = 0.7
PubMed Code: AAY89096
Description:
Isolated: 2005
Source: Taura syndrome virus
M1A2S3Y4Y5L6N7I8K9T10H11N12L13R14R15T16P17G18A19H20R21A22F23Y24V25M26N27D28D29G30E31N32R33I34Y35S36L37
I38G39T40L41R42R43A44P45A46F47K48V49G50S51R52R53Y54K55S56H57I58P59Y60R61R62K63A64T65V66A67E68L69C70
N71Q72L73H74D75R76V77L78P79F80A81N82P83Q84V85W86K87E88V89I90S91E92N93K94V95Q96P97D98S99M100L101K102
A103A104F105G106N107W108E109E110W111P112K113D114K115V116C117E118E119L120Y121S122E123C124E125C126G127
Y128V129G130T131C132Y133V134S135V136D137W138L139R140P141Q142A143T144K145C146N147D148C149I150L151K152
M153N154R155N156V157E158Y159P160Y161H162T163I164G165V166S167G168N169V170V171T172N173T174D175I176V177
Y178T179G180Y181A182D183V184F185K186C187E188K189C190D191L192L193M194G195A196W197A198P199N200D201I202
P203A204L205T206H207N208I209R210S211S212Q213C214V215Q216F217K218L219P220T221E222N223L224A225A226R227
N228Y229V230L231L232C233E234E235I236E237R238E239N240I241P242V243I244F245Q246D247Y248S249E250G251N252
V253F254T255C256R257I258V259S260G261D262L263T264A265V266G267T268A269S270N271M272Y273T274A275R276D277
V278A279S280K281S282L283L284D285Q286L287H288N289T290P291N292V293H294M295H296S297L298H299S300L301P302
Y303E304N305F306P307C308E309A310L311E312F313A314V315E316Q317G318I319I320P321P322V323T324F325D326E327
V328F329A330N331D332E333Y334V335I336T337I338S339C340S341L342L343V344V345S346D347V348G349P350T351Q352
A353V354A355R356E357R358A359A360K361R362F363L364K365M366Y367D368Y369S370A371S372Y373P374S375T376H377
M378F379T380L381S382T383L384P385Q386R387S388G389E390T391L392E393L394A395N396A397T398L399N400H401V402
N403N404V405I406D407R408H409D410E411A412I413S414N415V416R417Q418N419V420E421V422K423L424T425D426V427
S428R429Q430V431G432A433M434L435P436K437V438E439T440V441I442D443D444V445S446S447T448L449S450S451F452
R453G454V455L456D457K458I459S460A461W462M463P464S465S466N467P468K469I470I471D472L473I474K475E476T477
F478V479S480L481F482F483A484I485L486T487K488S489L490Y491P492I493I494Q495G496I497S498S499Y500A501L502
R503N504N505L506M507A508N509H510L511T512A513L514S515E516W517L518M519T520L521E522Y523D524S525P526D527
E528E529E530M531P532S533T534H535G536F537M538D539D540L541T542S543R544L545P546G547L548N549G550A551K552
V553Q554A555A556T557I558Y559E560S561I562G563T564G565L566C567V568A569L570S571G572I573L574S575F576I577
A578V579M580C581L582G583I584T585D586L587S588A589V590T591F592N593K594L595L596T597Q598S599S600L601V602
G603R604A605L606V607G608V609R610S611F612K613D614V615F616F617G618I619W620D621Y622V623D624N625Q626V627
C628E629I630L631Y632G633K634S635R636K637N638L639D640L641L642K643E644Y645P646S647L648D649S650L651L652
S653I654F655N656Y657F658H659D660T661V662D663A664N665V666L667I668S669C670N671R672A673A674C675E676L677
L678V679K680A681D682N683L684Y685Q686G687Y688L689D690K691S692I693T694L695M696H697R698E699I700S701S702
R703L704K705E706A707R708N709S710V711K712D713L714I715A716K717A718Q719V720Y721L722T723C724G725D726G727
S728R729V730P731P732V733V734V735Y736M737Y738G739D740A741G742C743G744K745T746E747L748S749M750A751L752
Q753D754H755F756A757T758K759Y760F761G762E763V764P765K766K767D768V769I770Y771S772R773K774A775E776N777
E778F779W780D781G782V783K784Q785S786H787K788I789I790A791Y792D793D794V795L796Q797I798V799D800S801A802
Q803K804P805N806P807E808L809F810E811F812I813R814L815N816N817S818D819P820Y821Q822V823H824M825S826S827
V828S829D830K831A832N833T834F835I836A837P838S839F840V841F842A843T844S845N846V847N848P849G850T851Y852
V853P854K855S856I857H858S859A860D861A862F863R864R865R866L867D868L869C870V871Y872V873D874V875K876D877
E878F879A880R881I882V883A884G885S886K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902
N903P904G905K906T907Q908H909D910M911K912H913E914I915V916A917G918T919Y920K921I922T923P924E925T926A927
V928Y929E930L931H932V933D934T935T936L937A938G939D940A941Q942S943K944V945C946A947Y948D949G950L951V952
S953L954I955E956Q957V958R959R960L961R962V963A964A965H966S967D968K969V970E971T972D973V974P975V976L977
P978T979R980L981H982E983L984S985Q986E987T988F989P990N991T992H993A994G995V996G997F998Q999F1000A1001T1002
D1003W1004L1005G1006D1007F1008D1009R1010P1011V1012E1013A1014L1015S1016Y1017L1018N1019K1020T1021L1022
E1023A1024H1025F1026V1027S1028R1029S1030A1031N1032D1033G1034S1035M1036F1037I1038P1039A1040S1041E1042
V1043A1044D1045L1046L1047C1048Q1049R1050H1051N1052N1053T1054N1055L1056N1057E1058E1059L1060V1061Y1062
L1063T1064W1065M1066T1067Q1068I1069T1070D1071K1072E1073L1074A1075S1076S1077L1078V1079Y1080F1081T1082
N1083N1084G1085M1086D1087K1088S1089I1090W1091K1092K1093S1094A1095E1096R1097S1098A1099Q1100A1101I1102
S1103Q1104C1105K1106N1107A1108W1109T1110R1111I1112N1113D1114F1115L1116K1117N1118H1119W1120I1121S1122
I1123S1124A1125V1126I1127G1128S1129A1130L1131L1132I1133G1134G1135V1136S1137S1138A1139V1140K1141C1142
A1143T1144K1145C1146R1147V1148R1149K1150I1151L1152Q1153D1154G1155G1156S1157I1158M1159Q1160L1161V1162
G1163V1164R1165S1166C1167M1168Y1169A1170C1171Q1172L1173C1174K1175R1176I1177K1178N1179C1180D1181L1182
R1183L1184R1185V1186R1187N1188R1189S1190E1191G1192V1193T1194T1195F1196V1197P1198G1199D1200V1201R1202
R1203V1204A1205R1206H1207V1208I1209S1210A1211A1212D1213V1214C1215E1216V1217P1218V1219H1220H1221S1222
F1223I1224Q1225S1226L1227C1228D1229E1230A1231F1232T1233V1234H1235S1236D1237K1238E1239E1240T1241F1242
S1243I1244L1245D1246F1247T1248P1249E1250A1251K1252G1253R1254N1255P1256P1257E1258S1259A1260V1261V1262
E1263S1264H1265Q1266D1267Y1268R1269V1270K1271A1272A1273V1274V1275E1276S1277H1278Q1279D1280F1281K1282
P1283K1284D1285A1286I1287V1288E1289S1290T1291I1292D1293T1294V1295F1296T1297E1298S1299H1300Q1301D1302
V1303R1304V1305K1306L1307H1308P1309Q1310I1311E1312S1313H1314Q1315D1316F1317R1318A1319K1320N1321P1322
I1323V1324E1325S1326R1327K1328P1329D1330Y1331Q1332V1333E1334W1335T1336D1337L1338R1339T1340E1341S1342
S1343N1344D1345R1346N1347A1348Q1349D1350I1351S1352N1353R1354I1355L1356S1357R1358N1359F1360V1361R1362
L1363Y1364V1365P1366G1367S1368S1369L1370Y1371T1372H1373G1374L1375F1376A1377Y1378G1379R1380M1381L1382
L1383M1384P1385K1386H1387M1388F1389D1390M1391L1392N1393G1394S1395V1396E1397I1398V1399S1400I1401A1402
D1403K1404G1405N1406T1407R1408V1409H1410V1411K1412I1413Q1414S1415H1416K1417T1418V1419T1420R1421G1422
G1423Y1424E1425V1426D1427I1428V1429I1430C1431E1432M1433G1434N1435S1436I1437S1438A1439R1440K1441D1442
I1443T1444S1445Y1446F1447P1448T1449V1450K1451E1452L1453P1454G1455L1456T1457G1458M1459M1460S1461S1462
G1463R1464M1465R1466V1467F1468S1469T1470A1471K1472F1473K1474A1475S1476D1477S1478C1479S1480Y1481L1482
M1483P1484Q1485D1486F1487V1488A1489K1490Y1491I1492A1493A1494V1495D1496H1497I1498T1499S1500K1501S1502
P1503E1504K1505K1506S1507Y1508F1509I1510R1511Q1512G1513F1514E1515A1516E1517S1518D1519S1520M1521Q1522
G1523D1524C1525C1526S1527P1528Y1529V1530L1531F1532N1533S1534A1535S1536R1537A1538K1539I1540V1541G1542
L1543H1544C1545A1546G1547F1548D1549G1550T1551A1552R1553V1554F1555A1556Q1557I1558I1559T1560Q1561E1562
D1563I1564M1565A1566A1567T1568P1569T1570T1571H1572A1573G1574R1575V1576T1577T1578E1579F1580P1581H1582
T1583S1584L1585R1586D1587S1588P1589L1590P1591N1592S1593M1594A1595I1596G1597S1598V1599K1600T1601A1602
P1603N1604P1605T1606K1607S1608E1609I1610T1611R1612S1613P1614I1615H1616G1617C1618F1619P1620V1621R1622
T1623A1624P1625A1626T1627L1628Y1629S1630P1631T1632E1633N1634L1635L1636I1637K1638N1639A1640M1641K1642
V1643T1644K1645N1646V1647E1648L1649L1650E1651E1652D1653L1654I1655D1656A1657C1658V1659H1660D1661V1662
K1663R1664I1665L1666N1667A1668P1669G1670V1671S1672D1673V1674E1675K1676R1677V1678L1679T1680H1681E1682
E1683S1684I1685T1686G1687I1688E1689N1690R1691Q1692Y1693M1694N1695A1696L1697N1698R1699S1700T1701S1702
A1703G1704F1705P1706Y1707S1708S1709R1710K1711A1712K1713G1714K1715S1716G1717K1718Q1719T1720W1721L1722
G1723S1724E1725E1726F1727I1728V1729D1730N1731P1732D1733L1734K1735E1736H1737V1738E1739K1740I1741V1742
D1743K1744A1745K1746D1747G1748I1749V1750D1751V1752S1753L1754G1755I1756F1757A1758A1759T1760L1761K1762
D1763E1764R1765R1766P1767L1768E1769K1770V1171Q1772A1773N1174K1775T1776R1777V1778F1779A1780A1781S1782
N1783Q1784G1785L1786A1787L1788A1789L1790R1791R1792Y1793Y1794L1795S1796F1797L1798D1799H1800V1801M1802
T1803N1804R1805I1806D1807N1808E1809I1810G1811L1812G1813V1814N1815V1816Y1817S1818Y1819D1820W1821T1822
R1823I1824V1825N1826K1827L1828K1829R1830V1831G1832D1833K1834V1835I1836A1837G1838D1839F1840S1841N1842
F1843D1844G1845S1846L1847N1848S1849Q1850I1851L1852S1853R1854V1855S1856E1857I1858V1859T1860D1861W1862
Y1863G1864D1865D1866A1867E1868N1869G1870L1871I1872R1873H1874T1875L1876L1877E1878Y1879L1880F1881N1882
A1883T1884W1885L1886M1887N1888G1889K1890V1891F1892Q1893L1894N1895H1896S1897Q1898P1899S1900G1901N1902
P1903L1904T1905T1906L1907I1908N1909C1910V1911Y1912N1913M1914I1915I1916F1917R1918Y1919V1920Y1921L1922
L1923A1924Q1925R1926E1927N1928G1929F1930P1931M1932T1933L1934S1935G1936F1937T1938T1939N1940V1941A1942
C1943I1944F1945Y1946G1947D1948D1949S1950L1951C1952S1953V1954S1955D1956K1957V1958S1959E1960W1961F1962
N1963Q1964H1965V1966I1967T1968R1969L1970M1971A1972A1973T1974G1975H1976E1977Y1978T1979D1980E1981T1982
K1983S1984G1985S1986P1987P1988P1989Y1990R1991S1992L1993N1994E1995V1996T1997F1998L1999K2000R2001E2002
F2003V2004L2005R2006D2007H2008F2009W2010I2011A2012P2013L2014S2015R2016N2017T2018I2019E2020D2021M2022
C2023M2024W2025S2026R2027K2028N2029I2030D2031A2032Q2033D2034A2035L2036L2037Q2038T2039T2040R2041I2042
A2043S2044F2045E2046A2047S2048L2049H2050E2051K2052N2053Y2054F2055L2056M2057F2058C2059D2060V2061I2062
K2063K2064A2065C2066R2067N2068A2069G2070Y2071K2072E2073A2074C2075L2076H2077E2078L2079D2080C2081K2082
S2083F2084L2085L2086A2087Q2088Q2089G2090R2091A2092G2093A2094H2095D2096S2097E2098F2099L2100S2101Q2102
L2103L2104D2105L2106N2107
(SEQ ID NO: 457)
Replikin Sequences in Amino-Terminal Portion of Peptide
(1) H20R21A22F23Y24V25M26N27D28D29G30E31N32R33I34Y35S36L37I38G39T40L41R42R43A44P45A46F47K48V49G50
S51R52R53Y54K55S56H57I58P59Y60R61R62K63
(SEQ ID NO: 458)
(2) K48V49G50S51R52R53Y54K55S56H57
(SEQ ID NO: 459)
(3) K48V49G50S51R52R53Y54K55S56H57I58P59Y60R61R62K63A64T65V66A67E68L69C70N71Q72L73H74
(SEQ ID NO: 460)
(4) K55S56H57I58P59Y60R61R62K63
(SEQ ID NO: 461)
(5) K55S56H57I58P59Y60R61R62K63A64T65V66A67E68L69C70N71Q72L73H74
(SEQ ID NO: 462)
(6) H57I58P59Y60R61R62K63A64T65V66A67E68L69C70N71Q72L73H74D75R76V77L78P79F80A81N82P83Q84V85W86K87
E88V89I90S91E92N93K94
(SEQ ID NO: 463)
(7) H57I58P59Y60R61R62K63A64T65V66A67E68L69C70N71Q72L73H74D75R76V77L78P79F80A81N82P83Q84V85W86K87
E88V89I90S91E92N93K94V95Q96P97D98S99M100L101K102
(SEQ ID NO: 464)
(8) H74D75R76V77L78P79F80A81N82P83Q84V85W86K87E88V89I90S91E92N93K94V95Q96P97D98S99M100L101K102
(SEQ ID NO: 465)
(9) H74D75R76V77L78P79F80A81N82P83Q84V85W86K87E88V89I90S91E92N93K94
(SEQ ID NO: 466)
(10) K145C146N147D148C149I150L151K152M153N154R155N156V157E158Y159P160Y161H162
(SEQ ID NO: 467)
(11) K469I470I471D472L473I474K475E476T477F478V479S480L481F482F483A484I485L486T487K488S489L490Y491P492
I493I494Q495G496I497S498S499Y500A501L502R503N504N505L506M507A508N509H510
(SEQ ID NO: 468)
(12) K634S635R636K637N638L639D640L641L642K643E644Y645P646S647L648D649S650L651L652S653I654F655N656Y657
F658H659
(SEQ ID NO: 469)
(13) K637N638L639D640L641L642K643E644Y645P646S647L648D649S650L651L652S653I654F655N656Y657F658H659
(SEQ ID NO: 470)
(14) H697R698E699I700S701S702R703L704K705E706A707R708N709S710V711K712
(SEQ ID NO: 471)
Replikin Sequences in Mid-Molecule Portion of Peptide
(15) K705E706A707R708N709S710V711K712D713L714I715A716K717A718Q719V720Y721L722T723C724G725D726G727S728
R729V730P731P732V733V734V735Y736M737Y738G739D740A741G742C743G744K745T746E747L748S749M750A751L752
Q753D754H755
(SEQ ID NO: 472)
(16) H755F756A757T758K759Y760F761G762E763V764P765K766
(SEQ ID NO: 473)
(17) H755F756A757T758K759Y760F761G762E763V764P765K766K767
(SEQ ID NO: 474)
(18) H755F756A757T758K759Y760F761G762E763V764P765K766K767D768V769I770Y771S772R773K774
(SEQ ID NO: 475)
(19) H755F756A757T758K759Y760F761G762E763V764P765K766K767D768V769I770Y771S772R773K774A775E776N777E778
F779W780D781G782V783K784
(SEQ ID NO: 476)
(20) K759Y760F761G762E763V764P765K766K767D768V769I770Y771S772R773K774A775E776N777E778F779W780D781G782
V783K784Q785S786H787
(SEQ ID NO: 477)
(21) K766K767D768V769I770Y771S772R773K774A775E776N777E778F779W780D781G782V783K784Q785S786H787
(SEQ ID NO: 478)
(22) K767D768V769I770Y771S772R773K774A775E776N777E778F779W780D781G782V783K784Q785S786H787
(SEQ ID NO: 479)
(23) K774A775E776N777E778F779W780D781G782V783K784Q785S786H787
(SEQ ID NO: 480)
(24) K774A775E776N777E778F779W780D781G782V783K784Q785S786H787K788I789I790A791Y792D793D794V795L796Q797
I798V799D800S801A802Q803K804P805N806P807E808L809F810E811F812I813R814L815N816N817S818D819P820Y821
Q822V823H824
(SEQ ID NO: 481)
(25) H858S859A860D861A862F863R864R865R866L867D868L869C870V871Y872V873D874V875K876D877E878F879A880R881
I882V883A884G885S886K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905
K906
(SEQ ID NO: 482)
(26) H858S859A860D861A862F863R864R865R866L867D868L869C870V871Y872V873D874V875K876D877E878F879A880R881
I882V883A884G885S886K887G888H889R890K891V892P893C894E895Q896K897
(SEQ ID NO: 483)
(27) K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910
M911K912H913E914I915V916A917G918T919Y920K921I922T923P924E925T926A927V928Y929E930L931H932
(SEQ ID NO: 484)
(28) K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901
(SEQ ID NO: 485)
(29) K887G888H889R890K891V892P893C894E895Q896K897
(SEQ ID NO: 486)
(30) K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909
(SEQ ID NO: 487)
(31) K887G888H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910
M911K912H913
(SEQ ID NO: 488)
(32) H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912
H913E914I915V916A917G918T919Y920K921
(SEQ ID NO: 489)
(33) H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906
(SEQ ID NO: 490)
(34) H889R890K891V892P893C894E895Q896K897
(SEQ ID NO: 491)
(35) H889R890K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912
(SEQ ID NO: 492)
(36) K891V892P893C894E895Q896K897I898W899L900H901
(SEQ ID NO: 493)
(37) K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909
(SEQ ID NO: 494)
(38) K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912H913
(SEQ ID NO: 495)
(39) K891V892P893C894E895Q896K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912H913E914
I915V916A917G918T919Y920K921I922T923P924E925T926A927V928Y929E930L931H932
(SEQ ID NO: 496)
(40) K897I898W899L900H901Q902N903P904G905K906
(SEQ ID NO: 497)
(41) K897I898W899L900H901Q902N903P904G905K906T907Q908H909
(SEQ ID NO: 498)
(42) K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912H913
(SEQ ID NO: 499)
(43) K897I898W899L900H901Q902N903P904G905K906T907Q908H909D910M911K912H913E914I915V916A917G918T919Y920
K921I922T923P924E925T926A927V928Y929E930L931H932
(SEQ ID NO: 500)
(44) H901Q902N903P904G905K906T907Q908H909D910M911K912
(SEQ ID NO: 501)
(45) H901Q902N903P904G905K906T907Q908H909D910M911K912H913E914I915V916A917G918T919Y920K921
(SEQ ID NO: 502)
(46) K906T907Q908H909D910M911K912
(SEQ ID NO: 503)
(47) K906T907Q908H909D910M911K912H913
(SEQ ID NO: 504)
(48) K906T907Q908H909D910M911K912H913E914I915V916A917G918T919Y920K921I922T923P924E925T926A927V928Y929
E930L931H932
(SEQ ID NO: 505)
(49) H909D910M911K912H913E914I915V916A917G918T919Y920K921
(SEQ ID NO: 506)
(59) K912H913E914I915V916A917G918T919Y920K921
(SEQ ID NO: 507)
(51) K912H913E914I915V916A917G918T919Y920K921I922T923P924E925T926A927V928Y929E930L931H932
(SEQ ID NO: 508)
(52) H1119W1120I1121S1122I1123S1124A1125V1126I1127G1128S1129A1130L1131L1132I1133G1134G1135V1136S1137
S1138A1139V1140K1141C1142A1143T1144K1145C1146R1147V1148R1149K1150
(SEQ ID NO: 509)
(53) H1278Q1279D1280F1281K1282P1283K1284D1285A1286I1287V1288E1289S1290T1291I1292D1293T1294V1295F1296
T1297E1298S1299H1300Q1301D1302V1303R1304V1305K1306L1307H1308P1309Q1310I1311E1312S1313H1314Q1315
D1316F1317R1318A1319K1320N1321P1322I1323V1324E1325S1326R1327K1328
(SEQ ID NO: 510)
(54) H1300Q1301D1302V1303R1304V1305K1306L1307H1308P1309Q1310I1311E1312S1313H1314Q1315D1316F1317R1318
A1319K1320N1321P1322I1323V1324E1325S1326R1327K1328
(SEQ ID NO: 511)
(55) H1308P1309Q1310I1311E1312S1313H1314Q1315D1316F1317R1318A1319K1320N1321P1322I1323V1324E1325S1326
R1327K1328
(SEQ ID NO: 512)
(56) H1314Q1315D1316F1317R1318A1319K1320N1321P1322I1323V1324E1325S1326R1327K1328
(SEQ ID NO: 513)
(57) H1373G1374L1375F1376A1377Y1378G1379R1380M1381L1382L1383M1384P1385K1386H1387M1388F1389D1390M1391
L1392N1393G1394S1395V1396E1397I1398V1399S1400I1401A1402D1403K1404G1405N1406T1407R1408V1409H1410
V1411K1412
(SEQ ID NO: 514)
(58) H1387M1388F1389D1390M1391L1392N1393G1394S1395V1396E1397I1398V1399S1400I1401A1402D1403K1404G1405
N1406T1407R1408V1409H1410V1411K1412
(SEQ ID NO: 515)
(59) K1404G1405N1406T1407R1408V1409H1410V1411K1412
(SEQ ID NO: 516)
(60) K1404G1405N1406T1407R1408V1409H1410V1411K1412I1413Q1414S1415H1416
(SEQ ID NO: 517)
Replikin Sequences in Carboxy-Terminal Portion of Peptide
(61) H1410V1411K1412I1413Q1414S1415H1416K1417T1418V1419T1420R1421G1422G1423Y1424E1425V1426D1427I1428
V1429I1430C1431E1432M1433G1434N1435S1436I1437S1438A1439R1440K1441D1442I1443T1444S1445Y1446F1447
P1448T1449V1450K1451
(SEQ ID NO: 518)
(62) H1416K1417T1418V1419T1420R1421G1422G1423Y1424E1425V1426D1427I1428V1429I1430C1431E1432M1433G1434
N1435S1436I1437S1438A1439R1440K1441D1442I1443T1444S1445Y1446F1447P1448T1449V1450K1451
(SEQ ID NO: 519)
(63) H1582T1583S1584L1585R1586D1587S1588P1589L1590P1591N1592S1593M1594A1595I1596G1597S1598V1599K1600
T1601A1602P1603N1604P1605T1606K1607
(SEQ ID NO: 520)
(64) K1600T1601A1602P1603N1604P1605T1606K1607S1608E1609I1610T1611R1612S1613P1614I1615H1616
(SEQ ID NO: 521)
(65) H1616G1617C1618F1619P1620V1621R1622T1623A1624P1625A1626T1627L1628Y1629S1630P1631T1632E1633N1634
L1635L1636I1637K1638N1639A1640M1641K1642V1643T1644K1645
(SEQ ID NO: 522)
(66) K1638N1639A1640M1641K1642V1643T1644K1645N1646V1647E1648L1649L1650E1651E1652D1653L1654I1655D1656
A1657C1658V1659H1660
(SEQ ID NO: 523)
(67) K1638N1639A1640M1641K1642V1643T1644K1645N1646V1647E1648L1649L1650E1651E1652D1653L1654I1655D1656
A1657C1658V1659H1660D1661V1662K1663R1664I1665L1666N1667A1668P1669G1670V1671S1672D1673V1674E1675
K1676R1677V1678L1679T1680H1681
(SEQ ID NO: 524)
(68) H1681E1682E1683S1684I1685T1686G1687I1688E1689N1690R1691Q1692Y1693M1694N1695A1696L1697N1698R1699
S1700T1701S1702A1703G1704F1705P1706Y1707S1708S1709R1710K1711A1712K1713G1714K1715S1716G1717K1718
(SEQ ID NO: 525)
(69) K1711A1712K1713G1714K1715S1716G1717K1718Q1719T1720W1721L1722G1723S1724E1725E1726F1727I1728V1729
D1730N1731P1732D1733L1734K1735E1736H1737
(SEQ ID NO: 526)
(70) K1735E1736H1737V1738E1739K1740I1741V1742D1743K1744
(SEQ ID NO: 527)
(71) H1737V1738E1739K1740I1741V1742D1743K1744A1745K1746D1747G1748I1749V1750D1751V1752S1753L1754G1755
I1756F1757A1758A1759T1760L1761K1762D1763E1764R1765R1766P1767L1768E1769K1770
(SEQ ID NO: 528)
(72) H1737V1738E1739K1740I1741V1742D1743K1744A1745K1746
(SEQ ID NO: 529)
(73) K1762D1763E1764R1765R1766P1767L1768E1769K1770V1771Q1772A1773N1774K1775T1776R1777V1778F1779A1780
A1781S1782N1783Q1784G1785L1786A1787L1788A1789L1790R1791R1792Y1793Y1794L1795S1796F1797L1798D1799
H1800
(SEQ ID NO: 530)
(74) H1800V1801M1802T1803N1804R1805I1806D1807N1808E1809I1810G1811L1812G1813V1814N1815V1816Y1817S1818
Y1819D1820W1821T1822R1823I1824V1825N1826K1827L1828K1829R1830V1831G1832D1833K1834
(SEQ ID NO: 531)
(75) K1827L1828K1829R1830V1831G1832D1833K1834V1835I1836A1837G1838D1839F1840S1841N1842F1843D1844G1845
S1846L1847N1848S1849Q1850I1851L1852S1853R1854V1855S1856E1857I1858V1859T1860D1861W1862Y1863G1864
D1865D1866A1867E1868N1869G1870L1871I1872R1873H1874
(SEQ ID NO: 532)
(76) H2050E2051K2052N2053Y2054F2055L2056M2057F2058C2059D2060V2061I2062K2063K2064A2065C2066R2067N2068
A2069G2070Y2071K2072
(SEQ ID NO: 533)
(77) H2050E2051K2052N2053Y2054F2055L2056M2057F2058C2059D2060V2061I2062K2063K2064A2065C2066R2067N2068
A2069G2070Y2071K2072E2073A2074C2075L2076H2077E2078L2079D2080C2081K2082
(SEQ ID NO: 534)
(78) K2063K2064A2065C2066R2067N2068A2069G2070Y2071K2072E2073A2074C2075L2076H2077
(SEQ ID NO: 535)
(79) K2063K2064A2065C2066R2067N2068A2069G2070Y2071K2072E2073A2074C2075L2076H2077E2078L2079D2080C2081
K2082S2083F2084L2085L2086A2087Q2088Q2089G2090R2091A2092G2093A2094H2095
(SEQ ID NO: 9)
(80) K2064A2065C2066R2067N2068A2069G2070Y2071K2072E2073A2074C2075L2076H2077E2078L2079D2080C2081K2082
S2083F2084L2085L2086A2087Q2088Q2089G2090R2091A2092G2093A2094H2095
(SEQ ID NO: 536)
(81) K2064A2065C2066R2067N2068A2069G2070Y2071K2072E2073A2074C2075L2076H2077
(SEQ ID NO: 537)
(82) K2072E2073A2074C2075L2076H2077E2078L2079D2080C2081K2082
(SEQ ID NO: 538)
(83) K2072E2073A2074C2075L2076H2077E2078L2079D2080C2081K2082S2083F2084L2085L2086A2087Q2088Q2089G2090
R2091A2092G2093A2094H2095
(SEQ ID NO: 539)
Replikin Count = Number of Replikins per 100 amino acids = 83/2107 = 3.9
Example 6 Determination of Replikin Concentration in Publicly Available Accession Numbers for Isolates of TSV from 2000 Through 2005
Mean Replikin concentrations were determined for all amino acid sequences for Taura Syndrome Virus with accession numbers publicly available at www.pubmed.com. The amino acid sequences were scanned for Replikin sequences of 7 to 50 amino acids comprising (1) at least one lysine residue located at a first terminus of the sequence and at least one lysine residue or at least one histidine residue located at a second terminus of the sequence; (2) a first lysine residue located six to ten residues from a second lysine residue; (3) at least one histidine residue; and (4) at least 6% lysine residues. The total number of Replikin sequences was determined for each available accession number. The total number of Replikin sequences in each accession number was then divided by the total number of amino acid residues disclosed in the accession number. The result was the Replikin concentration. The mean Replikin concentration was then determined for all viruses isolated and reported in a particular year. Table 5 provides the results.
TABLE 5
TSV Replikin Concentration by Year
No. of Mean Replikin
Isolates Concentration
Year PubMed Accession Number-Replikin Count per year per year S.D. Significance
2000 NP_149058 70 NP_149057 70 AAK72221 70 5 2.7 1.3 low p < 0.02
AAK72220 70 AAG44834 4
2001 AAM73766 7 1 0.7 0.0 prev p < 0.02
2002 AAN77089 2 AAN77088 2 AAN77087 2 8 0.7 0.4 low p > 0.50
AAN77086 2 AAW32934 2 AAW32932 2
AAW32930 2 AAW32929 1
2003 AAR11292 6 AAR11291 6 AAR11290 6 3 0.6 0.0 prev p < 0.20
2004 AAX07125 2 AAX07117 2 AAT81157 75 23 0.8 0.9 low p < 0.40,
AAT81158 75 AAX07127 2 AAX07126 2 prev p < 0.20
AAX07124 2 AAX07123 2 AAX07122 2
AAX07121 2 AAX07120 2 AAX07119 2
AAX07118 2 AAX07116 2 AAX07115 2
AAX07114 2 AAX07113 2 AAX07112 2
AAX35819 2 AAX35818 1 AAX35817 2
AAX35816 1 AAX35815 2
2005 AAY56364 71 AAY56363 71 AAY44822 1 12 1.8 1.7 low p < 0.02,
AAY44821 1 AAY44820 1 AAY44819 1 prev p < 0.05
AAY44818 1 AAY44817 1 AAY89097 83
AAY89096 83 ABB17263 63 ABB17264 63
Example 7 Comparison of the Replikin Concentration of Four Strains of Taura Syndrome Virus by an Independent Laboratory
The Replikin concentrations of the protein sequences of four Taura Syndrome Virus (TSV) isolates from Hawaii, Belize, Thailand and Venezuela, respectively, were examined without any knowledge of the virulence of the four isolates, and the virulence was ranked quantitatively in the order of the Replikin concentrations. The virulence of the four TSV isolates was compared in an independent laboratory, without any knowledge of the Replikin concentrations. The virulence was compared through a per os laboratory infection in juvenile Litopenaeus vannamei (Kona stock, Oceanic Institute, Hawaii). The results showed that the Belize isolate is the most virulent, the Thailand isolate is the second most virulent, followed by the Hawaii isolate, and the Venezuela isolate, which is the least virulent. This is based on the analyses of cumulative survivals at the end of a bioassay and based on the time of 50% mortality. TSV infection as the cause of death was confirmed by positive reactions in RT-PCR detection and by the appearance of characteristic lesions observed in histological analysis. The correlation of Replikin concentrations with virulence as indicated by the Mortality Rate was quantitative and linear.
Challenge Methods:
Small juveniles of specific-pathogen-free Litopenaeus vannamei (20 shrimp per tank, mean weight: 1.8 g) were fed minced TSV-infected tissues (infected separately with each of the 4 isolates originating from Belize, Thailand, Venezuela and Hawaii) for 3 days at 5% of their body weight. These shrimp were maintained with pelleted ration (Rangen 35%) for the following 12 days. Each challenge bioassay of a specific isolate was triplicated. During the bioassay period, all tanks were checked daily for dead or moribund shrimp. All mortalities were removed from the tank and frozen. One to three moribund shrimp from each isolate were preserved in Davidson's AFA fixative and processed for routine histology to confirm viral infection. For each isolate, six moribund shrimp were collected during the acute phase infection and total RNA was extracted from their gill tissues with a High Pure RNA tissue kit (Roche). The extracted RNA was analyzed for the presence of TSV by real-time RT-PCR.
All tanks were outfitted with an acclimated biological filter and aeration, and were covered with plastic to contain aerosols. The average salinity of the water was 23 ppt and the water temperature was 28° C. The challenge study was terminated after 15 days with live animals counted as survivors.
Results
Comparison of Virulence: Mortality in Shrimp
First mortality was seen on day 2 after exposure to TSV in all 4 isolates. For Belize isolate, most (83%) of shrimp died by day 4 and had a 0% survival at day 11 (FIG. 7, Table 6). For Thailand isolate, 63% mortalities occurred by day 4 and had a 20% survival at the end of 15-day bioassay (FIG. 7, Table 6). For Hawaii isolate, mortalities increased starting at day 2 and reached to a peak at day 5; the cumulative survival was 22% at the end (FIG. 7, Table 6). For Venezuela isolate, mortalities occurred slowly at days 2 and 3 with 22% of shrimp showed mortalities on day 4 and then mortalities slowed down; with 42% of shrimp surviving at the end (FIG. 7, Table 6). The time period for reaching 50% mortality caused by TSV infection for the isolate of Belize, Thailand, Hawaii and Venezuela were 2.8, 3.5, 4.5 and 7 days, respectively (Table 6).
TABLE 6
Results from per os TSV challenge in
SPF Litopenaeus vannamei (Kona stock)
GenBank No. Survival Day of 50%
TSV isolate (ORF1) (%)(Mean) mortality
Belize AAT81157 0 2.8
Thailand AAY56363 20 3.5
US-Hawaii AAK72220 22 4.5
Venezuela ABB17263 42 7.0*
*High variation was observed in Venezuela's triplicate tanks, thus the Day of 50% mortality was determined by Kaplan-Meier survival analysis with the Statistix 8 program.

Pathology
Histological analysis of the samples of L. vannamei juveniles is summarized in Table 7.
TABLE 7
Summary of histological findings
TSV Days after TSV
UAZ ID# Isolate exposure lesions1 LOS2
O6-407J/1 Belize 3 G4 G4
06-407F/1 Thailand 3 G4 G2
06-407D/1 Thailand 4 G4 G3
06-407E/1 Thailand 4 G3 G2
06-407A/1 Hawaii 4 G2 G3
06-407C/1 Hawaii 4 G2 G4
06-407H/1 Venezuela 4 G4 G2
Severity grade: G1: sign of infection; G2: moderate signs of infection; G3: moderate to high signs of infection; G4: severe infection.
1TSV lesions = Presence of TSV pathognomonic lesions in the gills, mouth, stomach, intecumental cuticular epithelium, and appendages.
2LOS = presence of lymphoid organ spheroids within the lymphoid organ.
Belize TSV. Acute lesions of diagnostic TSV infection were found in one representative shrimp sample at a severity grade of G4. Nuclear pyknosis and karyorrhexis were observed in the cuticular epithelium of the general body surface, appendages, gills, stomach and esophagus. Lymphoid organ spheroids were also found at severity grade G4.
Thailand TSV. Severe (G4) TSV infection was detected in 2 out of 3 shrimp, and another shrimp showed a moderate to high grade (G3) of infection. Lymphoid organ spheroids were found at severities of G2 and G3.
Hawaii TSV. Moderate level (G2) of TSV infection was detected in 2 shrimp collected at day 4. Lymphoid organ spheroids were found at severities of G3 and G4.
Venezuela TSV. Severe (G4) TSV infection was detected in one representative shrimp sampled at day 4. Lymphoid organ spheroids were found at severity of G2.
Real-Time TSV RT-PCR
All 24 samples (6 from each isolates) were all positive for TSV infection. This confirms that the mortalities observed from bioassays are from TSV infection.
TABLE 8
Mean and range of TSV RNA in gills
from shrimp challenge with TSV
TSV isolate Mean (Range) TSV copies/:l RNA
Belize 2.7 × 106 (4.8 × 105-4.4 × 106)
Thailand 2.7 × 106 (4.3 × 105-7.5 × 106)
Hawaii 5.2 × 107 (2.3 × 107-7.5 × 107)
Venezuela 6.5 × 105 (6.5 × 102-2.0 × 105)
TABLE 9
Percent Mortality and Blind Replikin Concentration
The results of 4 TSV virulence (percent mortality)
comparisons with blind Replikin Count are:
Isolate Percent Mortality Blind Replikin Concentration
Belize 100 3.5
Thailand 80 3.4
Hawaii 78.3 3.3
Venezuela 58.3 3.0
The order of virulence: Belize>Thailand> (or =) Hawaii>Venezuela, is in agreement with the Replikin concentration. The differences in the Replikin concentrations appear to be small but they are statistically significant at a level of p<0.001. See FIGS. 5 and 6.
Example 8 Increased Host Resistance to Taura Syndrome Virus by Administration of Synthetic Replikins
Shrimp cultured using the Challenge Methods described in Example 7 above were exposed in a first experiment for two weeks to synthetic Replikins per os mixed in their feed. The Replikins were peptides specific to Replikin sequences present in the TSV Hawaii strain isolate with which the shrimp were challenged/
In the experiment, mortality was reduced by 50% compared to a control group. The control group was given feed not containing synthetic Replikin sequences. A second control group was fed Replikin sequences synthesized with the covalent binding of additional amino acids to the same synthetic Replikins fed to the shrimp. The covalently “blocked” Replikins did not increase shrimp resistance to the virus in the same experiment demonstrating that the increase in host resistance was specific to the Replikin peptide structure.
Because little is known about the details of the immune system of the shrimp (shrimp appear not to produce antibodies), the phenomenon of “resistance” to infection appears to be based in a “primitive immune system” perhaps similar to the “toll receptor” and related systems. Thus the term “increased resistance” is used for the observed phenomenon and Replikin feed is used rather than “vaccine” for the administered substance which increases resistance.
The surviving shrimp of the first challenge were then set up in a fresh culture, fed for an additional two weeks with feed containing Replikin sequences, then again challenged with the Hawaii strain of Taura syndrome virus. The Replikin sequence supplemented feed was maintained while the survivors were again challenged repeatedly by the same virus, in repeated cycles, until 100% of the shrimp survived the TSV challenge.
Example 9 H5N1 Replikin Concentration and Mortality Determinations
The inventors analyzed and compared percent human mortality from H5N1 infections in years 2005 through the first quarter of 2007 to mean concentration of Replikin sequences in (1) the whole genome (2) the polymerase gene, (3) the pB1 gene area, (4) the pB2 gene area, and (5) the pA gene area, respectively, of H5N1 influenza strains isolated in 2003 through the first quarter of 2007. The following data were observed:
TABLE 10
H5N1 Replikin Concentration and Human Mortality
2003 2004 2005 2006 2007
H5N1 (Whole Virus) Replikin   2.2 +/− 1.2 2.4 +/− 1.4 2.3 +/− 2.6 3.8 +/− 4.6 3.7 +/− 4.5
Concentration
in Humans
H5N1 (Polymerase)   2.6 +/− 0.8 2.9 +/− 0.9 4.8 +/− 5.0 7.4 +/− 7.0 7.3 +/− 6.7
Replikin Concentration
In Humans
H5N1 2.0 +/− 0 2.0 +/− 0.1 8.0 +/7.7  16.1 +/− 5.7  15.4 +/− 5.9 
Replikin Peak Gene (pB1
gene area) Replikin
Concentration
in Humans
H5N1 pB2 gene area Replikin 2.4 +/− 0 2.8 +/− 0.3 2.4 +/0.4  2.4 +/− 0.1 2.4 +/− 0.3
Concentration
In Humans
H5N1 pA gene area Replikin 3.8 +/− 0 4.0 +/− 0.6 3.8 +/− 0.4 3.8 +/− 0.3 4.2 +/− 0.3
Concentration
In Humans
H5N1 Human Mortality 45 69 85
percent
Table 10 provides mortality data for H5N1 infections from 2005 through 2007 and does not include earlier mortality data. Mortality data prior to 2005 has not been included in Table 10 (or in the data in FIGS. 1-3) because data prior to 2005 is inconsistent and understood by those of skill in the art to contain errors, including errors caused by under-reporting. The first generally agreed occasion when there were human deaths caused by proven H5N1 infection was in Hong Kong in 1997-1998. (This is probably incorrect, however, since there probably was mortality between 1959, when H5N1 was first reported, and 1997). The usual figures cited for 1997 are: 30 human cases, 8 deaths with mortality rate of about 27%. The number of cases (morbidity) and the number who died (mortality) that were not reported is unknown, but suspected to be significant. These errors are usually high in geographic areas where the medical care is less structured and scientific, and the reporting is incomplete. Press reports between 1998 and 2002 were few, scattered, and not in agreement. Mortality data between 2005 and 2007 appear to be more consistent and have a higher level of reliability. Table 10, therefore, contains data from these years.
In Table 10, a correlation was established between mean human mortality and (1) mean concentration of Replikin sequences in the whole genome (two-fold increase from 2003 to 2007), (2) mean concentration of Replikin sequences in the polymerase gene (four-fold increase from 2003 to 2007) and (3) mean concentration of Replikin sequences in the Replikin Peak Gene (pB1 gene area) (eight-fold increase from 2003 to 2007) of H5N1 influenza strains. As Replikin concentration increased by these three measures, human mortality was observed to increase. See FIG. 16.
Over the same period (2003-2007), however, no significant increase was observed in the pB2 and pA gene areas. See FIG. 17. The pB2 and pA gene areas are neighbors to the Replikin Peak Gene found in the pB1 gene area. The observation that no significant increase occurred in the pB2 and pA gene areas during a remarkable increase in Replikin concentration in the neighboring pB1 area and a correlated significant increase in human mortality, provides support both for the significant capacity of Replikin concentration in the Replikin Peak Gene to predict virulence and for the precision of the method of predicting virulence using Replikin concentration in the Replikin Peak Gene Area.
As may be seen from Table 10, while the three measures (whole genome Replikin concentration, polymerase gene Replikin concentration, and pB1 gene area Peak Gene Replikin concentration) provided a correlation with human mortality, changes in the Replikin concentration in the polymerase gene correlated more significantly with human mortality, and changes in the Replikin concentration in the Replikin Peak Gene (pB1 gene area) of the H5N1 genome correlated still more significantly with human mortality. Table 10 demonstrates, therefore, that identification of Replikin Peak Genes within viral genomes improves identifications and predictions of virulence and mechansisms of virulence using Replikin concentration data. As seen in Table 10, the increase in Replikin concentration is magnified in its correlation with human mortality when restricted to changes in Replikin concentration in the polymerase genes and magnified still when restricted solely to the Replikin Peak Gene identified using the methods described herein.
Test of Reliability of Method of Predicting Outbreaks with Replikin Concentration
In addition to the correlative aspect of the increase in Replikin concentration being related to percent mortality, the data in Table 10 provides strong confirmation of the power and validity of the methodology of predicting changes in virulence and outbreaks of virus with changes in Replikin concentration. These data represent an objective test of the method of independently selecting and examining several thousand individual accession numbers within approximately 12 million total accession numbers in PubMed wherein each selection is independently submitted to the PubMed data base under a separate request using objective software. If there were not a reliable principle and a reliable method underlying each request, the potential for obtaining random results, or no results, or results which do not track each other at p<0.001 would markedly increase. Table 10 provides results wherein p was less than 0.001 between each group as compared one to another.
In Table 10 the structures that are correlated have, to the knowledge of the inventors, not been correlated before, that is, the inventors have examined the relationship of one internal virus structure to another internal virus structure or structures (e.g., three-way relationship between whole virus gene area, polymerase, and Replikin Peak Gene area) and have examined the external relation of these two or more internal structures to a host result of the virus infection, that is, percent mortality.
Table 10 represents consistent reproducible data, on repeated trials, which is the essence of the reliability of any method. For example, Table 10 provides independent data on (1) whole virus concentration of Replikins, (2) only the polymerase concentration of Replikins, and (3) only the Replikin Peak Gene concentration of Replikins. The data is then correlated with H5N1 mortality three times, namely in 2005, 2006 and 2007. The absence of significant changes in the pA and pB2 gene areas provides a control. In each case, the method measures Replikin concentration three ways, each of which correctly predict mortality, independently, thereby confirming the method, and further illustrating in the process, the magnifying function of the Replikin Peak Gene.
The sequence listing, saved as a filed named 47501-seqlisting.txt, created on Dec. 20, 2007, and totaling 241,027 bytes, is hereby incorporated by reference in its entirety.

Claims (21)

1. A method of determining an increased probability of an outbreak of influenza virus within about one to about three years following an increase in Replikin concentration in an isolate of White Spot Syndrome Virus or an isolate of Taura Syndrome Virus comprising identifying said increase in the concentration of Replikin sequences in at least one first isolate of White Spot Syndrome Virus or Taura Syndrome Virus as compared to at least one other isolate of White Spot Syndrome Virus or Taura Syndrome Virus, wherein said at least one first isolate is isolated at least six months later than said at least one other isolate is isolated, and wherein when said at least one first isolate is a White Spot Syndrome Virus isolate said at least one other isolate is a White Spot Syndrome Virus isolate, and when said at least one first isolate is a Taura Syndrome Virus isolate said at least one other isolate is a Taura Syndrome Virus isolate, and wherein said increase in the concentration of Replikin sequences signifies the increased probability of the outbreak of influenza virus within about one to about three years following said increase in the concentration of Replikin sequences, wherein said method is performed on a processor.
2. The method of claim 1 wherein said identification of an increase in Replikin concentration in said at least one first isolate comprises: determining the concentration of Replikin sequences in (i) a plurality of isolates of White Spot Syndrome Virus, wherein said at least one first isolate has been isolated about six months to about three years later than said at least one other of said isolates of White Spot Syndrome Virus, or (ii) a plurality of isolates of Taura Syndrome Virus, wherein said at least one first isolate of Taura Syndrome Virus is isolated about six months to about three years later than at least one other isolate of Taura Syndrome Virus.
3. A computer readable medium having instructions stored thereon, wherein executing said instructions comprises
using a processor to perform a method for predicting an outbreak of influenza virus comprising:
(1) determining a Replikin concentration of amino acid or nucleic acid sequences of a plurality of isolates of a virus wherein said virus is present in a reservoir of influenza virus and wherein at least one first isolate has been isolated about six months to about three years later than at least one other isolate; and
(2) predicting an outbreak of influenza virus by identifying an increase in Replikin concentration in said at least one first isolate as compared to said at least one other isolate by comparing the Replikin concentrations of said at least one first isolate with said at least one other isolate wherein said outbreak of influenza virus is predicted to occur within about one to about three years from the time point at which said at least one first isolate was isolated.
4. The method of claim 1, wherein said increase in the concentration of Replikin sequences in said at least one first isolate as compared to said at least one other isolate is statistically significant.
5. The method of claim 1, wherein said increase in said concentration of Replikin sequences of said plurality of isolates is an increase in the mean concentration of at least two first isolates as compared to the mean concentration of at least two other isolates wherein said at least two first isolates are isolated about six months to about three years later than said at least two other isolates.
6. The method of claim 5, wherein said increase in said mean concentration of said at least two first isolates as compared to the mean concentration of said at least two other isolates is statistically significant.
7. The method of claim 1, wherein said increase in Replikin concentration is in an isolate of White Spot Syndrome Virus.
8. The method of claim 1, wherein said increase in Replikin concentration is in an isolate of Taura Syndrome Virus.
9. A method of predicting an outbreak of influenza virus comprising:
(1) obtaining a plurality of isolates of White Spot Syndrome Virus or a plurality of isolates of Taura Syndrome Virus wherein at least one of said isolates of White Spot Syndrome Virus is isolated about six months to about 3 years later than at least one other of said isolates of White Spot Syndrome Virus or wherein at least one of said isolates of Taura Syndrome Virus is isolated about six months to about 3 years later than at least one other of said isolates of Taura Syndrome Virus;
(2) analyzing the amino acid sequence of at least one encoded protein or protein fragment or a combination of encoded proteins and/or protein fragments in each isolate of the plurality of isolates of White Spot Syndrome Virus or the plurality of isolates of Taura Syndrome Virus for the presence and concentration of encoded Replikin sequences;
(3) comparing the concentrations of encoded Replikin sequences in the at least one protein or protein fragment or in the combination of proteins or protein fragments in each isolate of the plurality of isolates of White Spot Syndrome Virus or in each isolate of the plurality of isolates of Taura Syndrome Virus;
(4) identifying an increase in the concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus or in said plurality of isolates of Taura Syndrome Virus over at least one time period of about six months or greater; and
(5) predicting an outbreak of influenza within about one to about three years following said identified increase in the concentration of Replikin sequences in said isolates of White Spot Syndrome Virus or in the concentration of Replikin sequences in said isolates of Taura Syndrome Virus.
10. The method of claim 9, further comprising:
(1) identifying at least one Replikin Scaffold sequence present in at least one isolate of the plurality of isolates of White Spot Syndrome Virus identified as having an increase in the concentration of Replikin sequences as compared to at least one other isolate of the plurality of isolates of White Spot Syndrome Virus over a time period of about six months or greater or identifying at least one Replikin Scaffold sequence present in at least one isolate in the plurality of isolates of Taura Syndrome Virus identified as having an increase in the concentration of Replikin sequences as compared to at least one other isolate of the plurality of isolates of Taura Syndrome Virus over a time period of about six months or greater;
(2) identifying at least one Replikin Scaffold sequence present in at least one isolate of a strain of influenza virus; and
(3) predicting the strain of influenza virus in which the at least one Replikin Scaffold sequence is identified as the strain of virus that will cause the predicted outbreak of influenza virus wherein a Replikin Scaffold is a peptide consisting of about 16 to about 34 amino acids comprising (1) a terminal lysine and optionally a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and optionally a histidine immediately adjacent to the terminal histidine, (3) a lysine within about 6 to about 10 amino acids of another lysine; and (4) at least 6% lysines.
11. The method of claim 10, wherein the Replikin Scaffold is a peptide having about 27 to about 33 amino acids.
12. The method of claim 9, wherein the increase in concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus over at least one time period of about six months or more is ten or more Replikin sequences per 100 amino acids.
13. The method of claim 12, wherein the increase in concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus over at least one time period of about six months or more is 90 or more Replikin sequences per 100 amino acids.
14. The method of claim 9, wherein the increase in concentration of encoded Replikin sequences in said plurality of isolates of Taura Syndrome Virus over at least one time period of about six months or more is one or more Replikin sequences per 100 amino acids.
15. The method of claim 14, wherein the increase in concentration of encoded Replikin sequences in said plurality of isolates of Taura Syndrome Virus over at least one time period of about six months or more is 2 or more Replikin sequences per 100 amino acids.
16. The method of claim 9, further comprising identifying an increase in the concentration of encoded Replikin sequences in said plurality of isolates of White Spot Syndrome Virus or said plurality of isolates of Taura Syndrome Virus over at least one to about three years.
17. The method of claim 1, wherein said outbreak of influenza is an outbreak of influenza A virus.
18. The method of claim 17 wherein said outbreak of influenza A virus is an outbreak of an H5N1 strain of influenza virus.
19. A method of predicting an outbreak of influenza virus comprising:
(1) obtaining a plurality of isolates of a first virus from at least one reservoir of Replikin sequences shared by influenza virus wherein the reservoir of Replikin sequences shared by influenza virus is any source of Replikin sequences that may be shared with an influenza virus including any host of influenza virus, any food source of a host of the influenza virus, any vector of influenza virus, or any substance wherein the genetic information of the influenza virus may be shared, mingled, mixed, exchanged or come into the proximity of the Replikin sequences of the reservoir;
(2) analyzing the amino acid sequence of at least one encoded protein or protein fragment or a combination of encoded proteins and/or protein fragments in each isolate of the plurality of isolates of said first virus for the presence and concentration of encoded Replikin sequences;
(3) comparing the concentrations of encoded Replikin sequences in the at least one protein or protein fragment or in the combination of proteins or protein fragments in each isolate of the plurality of isolates of said first virus;
(4) identifying an increase in the concentration of encoded Replikin sequences in said plurality of isolates of said virus over at least one time period of about six months or greater; and
(5) predicting an outbreak of influenza within about one to about three years following said identified increase in the concentration of Replikin sequences in said isolates of said first virus.
20. The method of claim 19, further comprising:
(1) identifying at least one Replikin Scaffold sequence present in at least one isolate of the plurality of isolates of said first virus identified as having an increase in the concentration of Replikin sequences as compared to at least one other isolate of the plurality of isolates of said virus over a time period of about six month;
(2) identifying at least one Replikin Scaffold sequence present in at least one isolate of a strain of influenza virus; and
(3) predicting the strain of influenza virus in which the at least one Replikin Scaffold sequence is identified as the strain of virus that will cause the predicted outbreak of influenza virus wherein a Replikin Scaffold is a peptide consisting of about 16 to about 34 amino acids comprising (1) a terminal lysine and optionally a lysine immediately adjacent to the terminal lysine; (2) a terminal histidine and optionally a histidine immediately adjacent to the terminal histidine, (3) a lysine within about 6 to about 10 amino acids of another lysine; and (4) at least 6% lysines.
21. The method of claim 19 or claim 20, wherein the reservoir is selected from the group consisting of shrimp, bird, migratory bird, chicken, duck, goose, and domestic bird.
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